Antimicrobials and methods of use thereof

ABSTRACT

This disclosure provides generally for antimicrobial compositions and methods of use comprising an anthocyanin, an anthocyanidin or metabolites thereof. Methods for promoting healing of a wound using these compositions are also disclosed. These compositions have broad spectrum antimicrobial activity and are safe for human and animal uses. Further, these compositions are safe for medical uses and industrial uses as antiseptic preparations to reduce or prevent microbial growth, including killing bacterial biofilms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/728,760, filed Oct. 10, 2017, which is a divisional of U.S.application Ser. No. 15/484,827, filed Apr. 11, 2017, now U.S. Pat. No.10,016,380, issued Jul. 10, 2018, which is a continuation-in-part ofU.S. application Ser. No. 15/189,510, filed Jun. 22, 2016, now U.S. Pat.No. 9,925,152, issued Mar. 27, 2018, which is a divisional of U.S.application Ser. No. 14/264,553, filed Apr. 29, 2014, now U.S. Pat. No.9,498,413, issued Nov. 22, 2016, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 61/818,275, filedMay 1, 2013, the disclosures of which are incorporated, in theirentirety, by this reference.

FIELD OF THE INVENTION

The present invention generally relates to antimicrobial compositions,methods for promoting healing of a wound and more specifically tomethods and compositions including the administration of an anthocyaninor an anthocyanidin or metabolites thereof for promoting wound healingby reducing or preventing microbial growth and inducing the activationof growth hormones. Pharmaceutical and nutraceutical compositionscontaining anthocyanin or anthocyanidins or metabolites thereof suitablefor administration to a mammal for promoting or inducing wound healingare also described.

The inventions provided herein also relate to compositions and methodsof sanitizing (skin antiseptic) or sterilizing solid surfaces, porous orsemi-porous or cloth like surfaces, such as materials or cloth orbandages. Also provided herein are compositions and methods for killingbacterial biofilms, which may be present on implantable devices, solidsurfaces, porous or semi-porous, or cloth-like material.

BACKGROUND OF THE INVENTION

There are many illnesses and conditions which are effectively treated bythe application of suitable antimicrobial agents. Many microorganisms,however, are increasingly difficult to treat because of resistance orallergic reactions to current antimicrobial agents. The development ofresistance is due in part to overuse of the antibiotic and subsequentbacteria mutation. (Blaser, M. Antibiotic overuse: Stop the killing ofbeneficial bacteria Nature 476, 393-394 (25 Aug. 2011).

The Centers for Disease Control and Prevention (CDC) estimated at least2 million people in the United States become infected with bacteria thatare resistant to antibiotics and at least 23,000 people die each year asa direct result of these infections. (Antibiotic Resistance Threats inthe United States, 2013, Centers for Disease Control and Prevention,Atlanta, Ga., USA 2013). The CDC report classified three microorganismswith an antibiotic resistance threat level of urgent in the UnitedStates and twelve microorganisms with an antibiotic resistance threatlevel of serious. Specifically, Clostridium difficile,Carbapenem-resistant Enterobacteriacaeae (CRE) and drug resistantNeisseria gonorrhoeae (cephalosporin resistance) are classified by theCDC as urgent because they require urgent public health attention toidentify infections and to limit transmission. Of these, the CDC states“Clostridium difficile is the most frequent etiologic agent forhealth-care—associated diarrhea. In one hospital, 30% of adults whodeveloped health-care—associated diarrhea were positive for C.difficile. Risk factors for acquiring C. difficile-associated infectioninclude a) exposure to antibiotic therapy, particularly with beta-lactamagents; b) gastrointestinal procedures and surgery; c) advanced age; andd) indiscriminate use of antibiotics. Of all the measures that have beenused to prevent the spread of C. difficile-associated diarrhea, the mostsuccessful has been the restriction of the use of antimicrobial agents.”(Sehulster L, Centers for Disease Control and Prevention, Guidelines forenvironmental infection control in healthcare facilities. MMWR 2003;52(RR10); 1-42). C. difficile is an anaerobic, gram-positive bacteriumcapable of sporulating when environmental conditions no longer supportits continued growth. The capacity to form spores enables the organismto persist in the environment (e.g., on dry surfaces or in soil) forextended periods of time. Environmental contamination by thismicroorganism is well known, especially direct exposure to contaminatedpatient-care items and high-touch surfaces in patients' bathrooms havebeen implicated as sources of infection. The CDC stated, “More needs tobe done to prevent C. difficile infections (CDIs). Major reductions willrequire antibiotic stewardship along with infection control applied tonursing homes and ambulatory-care settings as well as hospitals. Statehealth departments and partner organizations have shown leadership inpreventing CDIs in hospitals and can prevent more CDIs by extendingtheir programs to cover other health-care settings.” (CDC, Vital Signs;Preventing Clostridium difficile infections, MMWR 2012; 61-157-62).Because C. difficile spores resist killing by usual hospitaldisinfectants, an Environmental Protection Agency—registereddisinfectant with a C. difficile sporicidal label claim should be usedto augment thorough physical cleaning.

Twelve serious antibiotic-resistant threats identified in the CDC reportinclude: multidrug-resistant Acinetobacter, Drug-resistantCampylobacter, Fluconazole-resistant Candida (fungus), Extended spectrumβ-lactamase producing Enterobacteriacaea (ESBLs), Vancomycin-resistantEnterococcus (VRE), Multidrug-resistant Psuedomonas Aeruginosa,Drug-resistant Non-typhoidal Salmonella, Drug-resistant SalmonellaTyphi, Drug-resistant Shigella, Methicillin-resistant Staphylococcusauereus (MRSA), Drug-resistant Streptococcus pneumonia, Drug-resistanttuberculosis (MDR and XDR)(Antibiotic Resistance Threats in the UnitedStates, 2013, Centers for Disease Control and Prevention, Atlanta, Ga.,USA 2013). Of the twelve serious antibiotic-resistant threats identifiedin the CDC report, Methicillin-resistant Staphylococcus aureus (MRSA) isthe most frequently identified antimicrobial drug-resistant pathogen inU.S. hospitals. MRSA was one of the first pathogens to developresistance, first detected the United Kingdom in 1961. In 1999, MRSA wasresponsible for 37% of fatal cases of sepsis in the UK. Additionally,half of all S. aureus infections in the U.S. are resistant topenicillin, methicillin, tetracycline and erythromycin, leaving onlyvancomycin as an effective agent against S. aureus; however, strainswith intermediate levels of resistance, termed glycopeptide-intermediateStaphylococcus aureus (GISA) or vancomycin-intermediate Staphylococcusaureus (VISA), began appearing in the late 1990s and oxazolidinone,(linezolid) resistance in S. aureus was reported in 2001. Additionally,community-acquired MRSA (CA-MRSA) has now emerged as an epidemic that isresponsible for rapidly progressive, fatal diseases, includingnecrotizing pneumonia, sepsis, and necrotizing fasciitis. Outbreaks ofCA-MRSA infections have been reported in correctional facilities, amongathletic teams and military recruits, and in nurseries.

In addition to resistance, current antibiotics also have a limited usedue to allergic reactions in many patients (Romano A, Caubet J C.Antibiotic allergies in children and adults: from clinical symptoms toskin testing diagnosis. J Allergy Clin Immunol Pract. 2014January-February; 2(1):3-12).

Not only does resistance and allergic reactions to current antimicrobialagents result in increased patient morbidity and even mortality, butineffectiveness of current antimicrobial agents is also a major expenseto society. Surgical infections are costly not only because of cost oftreatment, including potentially hospitalization, but also the loss ofproductive work. This is exemplified in treatment of an infected totalknee replacement. The relative incidence of operative infections wasreported as 2.0% and 2.4% following total knee surgery. The most commoncause of revision total knee surgery (25.2%) is infection. (Bozic K J etal. The epidemiology of revision total knee arthroplasty in the UnitedStates. Clin Orthop Relat Res. 2010 January; 468(1):45-51). The annualcost of infected revisions to U.S. hospitals increased from $320 millionto $566 million during the study period and was projected to exceed$1.62 billion by 2020. (Kurtz S M, et al Economic burden ofperiprosthetic joint infection in the United States. J Arthroplasty.2012 September; 27(8 Suppl) The average cost of the surgical revision ofan infected total knee replacement was $116,383 in the years 2001through 2007. (Kapadia B H, et al. The Economic Impact of PeriprostheticInfections Following Total Knee Arthroplasty at a SpecializedTertiary-Care Center. J Arthroplasty. 2013 Oct. 17).

There is also a high cost to prophylactic antibiotic treatment prior toand around the time of surgery. (Chaweewannakom U. et al., Cost analysisof peri-operative antibiotic administration in total knee arthroplasty.J Med Assoc Thai. 2012 October; 95 Suppl 10:S42-7; Hebert C K, et al.,Cost of treating an infected total knee replacement. Clin Orthop RelatRes. 1996 October; (331):140-5). Furthermore, these costs may not becovered by government insurance in the U.S. especially with the largepersonal deductibles people have chosen under the Affordable Care Act.This problem already exists in Germany. This is a burden on the patientif they pay. If they do not pay then the cost is shifted to the doctorand the hospital. (Haenle M. et al. Economic impact of infected totalknee arthroplasty. Scientific World Journal. 2012; 2012:1 96515).

Treatment of the failed infected total joint may include repeat surgery,removal of the implant, insertion of an antibiotic impregnated spacer,hospitalization, therapy and return at later date to remove spacer andredo the total joint with hospitalization and long term antibiotics.(Garg P, et al. Antibiotic-impregnated articulating cement spacer forinfected total knee arthroplasty. Indian J Orthop. 2011 November;45(6):535-540).

Fungal infections also are problematic and have become less susceptibleto current antimicrobial agents. In hospitalized patients, fungalinfections are the fourth common cause of blood stream infection.Candida albicans is the major fungal pathogen of humans. (Warren, N G,American Society for Microbiology; 1995. 723; Bachmann, S P,Antimicrobial Agents Chemother, 2002; 46: 3591). It has been reportedthat mortality rate of patients with catheter related candidemiaapproaches 40%. (Fux, C A, Trends Microbiol, 2005; 13(1): 34; andTampakakis, E., Eukaryot Cell, 2009; 8:732). Biofilms of C. albicans arecapable of holding other micro-organisms and more likely to beheterogeneous with other bacteria and fungi in the environment and onmedical devices. (E Tam pakakis, A Y Peleg, E Mylonakis. Eukaryot Cell,2009; 8:732.) Moreover, biofilm cells are significantly less susceptibleto antimicrobial agents. As a result, drug therapy for an implantinfection may be futile, and often, the only solution is mechanicalremoval of the implant. (Melissa J J, et al, Antimicrob AgentsChemother. 2009; 53(6): 2638; and Anderson, J B, Nat Rev Microbiol,2005; 3(7): 547). Biofilm formation also plays an important role inoutbreaks of C. albicans related infections.

Between 1935 and 1968, 14 different classes of antibiotic weredeveloped. In the 45 years since then, only five have been brought out.No new classes have now been developed since 1987 (last 30 years).Because of this lack of new antibiotics, and because of the acquiredresistance to the known antibiotics, there is a continuing need for newantimicrobials and compositions, which are effective in reducing orpreventing microorganism growth. The new antimicrobials and compositionscan have a broad spectrum of utility without a history of overuse orresistance. The new methods and compositions can be applicable topromoting healing of wounds. In addition, the antimicrobials andcompositions would have no known allergic manifestation. Further the newantimicrobials and compositions would be cost effective. There is also aneed for antimicrobial compositions that can be effectively used onvarious surfaces, including high-touch surfaces such as light switchesand bathroom fixtures, medical devices, patient-care items, and the liketo reduce microbial contamination.

At present, in order to minimize the incidence of perisurgical woundinfections the patient is given preoperative antibiotics, intraoperative intra venous antibiotic, intraoperative antibiotic woundirrigation. (Heller S, Rezapoor M, Parvizi J. Minimising the risk ofinfection: a peri-operative checklist. Bone Joint J. 2016 January;98-B(1 Suppl A):18-22.); (Whiteside L A. Prophylactic peri-operativelocal antibiotic irrigation. Bone Joint J. 2016 January; 98-B(1 SupplA):23-6.)

Often antibiotic crystals and/or powder are placed in about the implantand the wound the wound before closure. (Bakhsheshian J, Dandaleh N S,Lam S K, Savage J W, Smith Z A. The use of vancomycin powder in modernspine surgery: systematic review and meta-analysis of the clinicalevidence. World Neurosurg. 2015 May; 83(5):816-23); (Molinari R W, KheraO A, Molinari III W J. Prophylactic intraoperative powdered vancomycinand postoperative deep spinal wound infection: 1,512 consecutivesurgical cases over a 6-year period. Eur Spine J. 2012 June; 21(Suppl4): 476-482.)

Attempts have been made by major implant OEM's to coat implants with anantibiotic at time of manufacture. (personal communication fromSmith+Nephew Company) but without success. The attempts have beenabandoned at the time of this application due to lack of a method andthe high barrier cost of 50 to 150 million dollars to perhaps gain FDAapproval. In spite of all these measures, peri surgical infections stilloccur. There is a need for effective means of reducing peri surgicalinfections following implant surgery, especially ones that at prone tobiofilms formation.

Previous clinical studies performed with various skin surfacedisinfectants have not been successful in decreasing the presence ofPropionibacterium acnes. The reason is that the regents used were notaccompanied by a vehicle that would penetrate intact human skin. (Lee MJ, Pottinger P S, Butler-Wu S, Bumgarne R E, Russ S M, Mattsenlll F A.Propionibacterium Persists in the Skin Despite Standard SurgicalPreparation. J Bone Joint Surg Am, 2014 Sep. 3; 96 (17): 1447-1450);(Saltzman M D, Nuber G W, Gryzlo S M, Mareck G S, Koh J L. Efficacy ofSurgical Preparation Solutions in Shoulder Surgery. J Bone Joint SurgAm, 2009 Aug. 1; 91 (8): 1949-1953); (McLellan E, Rownsend R, Parsons HK. Evaluation of ChloraPrep (2% chlorhexidine gluconate in 70% isopropylalcohol) for skin antiseptic in preparation for blood culturecollection. Journal of Infection, Volume 57, Issue 6, 459-463). This islikely due to the fact that P. acnes normally reside deep in the skinsurface within the hair follicles and/or sebaceous glands. Therefore, itnecessary to have a composition of matter in treating potentialpathogens on the human skin, especially P. acnes that contains withinthe vehicle a property than enhances skin penetration. Nakatsuji et alshowed that the microbiota extends within the dermis, therefore,enabling physical contact between bacteria and various cells below thebasement membrane. These observations show that normal commensalbacterial communities directly communicate with the host in a tissuepreviously thought to be sterile. (Nakatsuji T, Chiang H I, Jiang S B,Nagarajan H, Zengler K, Gallo R L. The microbiome extends tosubepidermal compartments of normal skin. Nat Commun. 2013; 4:1431.)Zeeuwen et. al. showed the bacterial communities of the surface of humanskin, mostly under static conditions in healthy volunteers differs fromwhat is found following skin injury. The dynamics of re-colonization ofskin microbiota following skin barrier disruption by tape stripping as amodel of superficial injury showed microbiome of the deeper layers,rather than that of the superficial skin layer, may be regarded as thehost indigenous microbiome. (Zeeuwen P L, Boekhorst J, van den Bogaard EH, de Koning H D, van de Kerkhof P M, Saulnier D M, van Swam II, vanHijum S A, Kleerebezem M, Schalkwijk J, Timmerman H M. Microbiomedynamics of human epidermis following skin barrier disruption. GenomeBiol. 2012 Nov. 15; 13(11):R101.) Therefore, there is a need for acomposition that has skin penetration properties to deliver an effectiveskin antiseptic regent and there remains a need for a composition usefulin treating potential pathogens on the human skin, especially P. acnesthat contains within the composition a substances than enhances skinpenetration.

Donlan's comprehensive review of biofilms reported that when antibioticswere first developed, the pathologic bacteria were singular and freefloating. They were characterized as planktonic. Subsequently bacteriahave developed resistance by mutation and biofilms formation.Leeuwenhoek first observed a biofilm is an assemblage ofsurface-associated microbial cells that is enclosed in an extracellularpolymeric substance matrix. (Donlan R M. Biofilms: Microbial Life onSurfaces. Emerg Infect Disease. 2002; 8(9):881-890). In addition,Donlan's review stated that biofilms are highly resistant to mostantimicrobial agents and disinfectants (Donlan R M. Role of biofilms inantimicrobial resistance. ASAIO J. 2000; 46:S47-52.). In addition,organisms within biofilms can readily acquire resistance through thetransfer of resistance plasmids. Such resistance could be especiallyacute in the health-care environment for patients with colonized urinarycatheters and collection bags. Many of the enteric organisms shown tocolonize urinary catheters carry plasmids encoding resistance tomultiple antimicrobial agents (Sedor J, Mulholland S G. Hospitalacquired urinary tract infections associated with the indwellingcatheter. Urol Clin North Am. 1999; 26:821-8).

Transfer of plasmids within biofilms has been well established (asalready discussed). Resistant organisms such as methicillin-resistantStaphylococcus aureus have also been shown to form biofilms (Murga R,McAllister S, Miller J M, Tenover F, Bell M, Donlan R M. Effect ofvancomycin treatment of methicillin-resistant S. aureus (MRSA) biofilmson central venous catheters in a model system. Poster No. C276 presentedat the 2001 American Society for Microbiology Annual Meeting, Orlando,Fla., May 23, 2001.) Biofilms may form on a wide variety of surfaces,including living tissues, indwelling medical devices, industrial orpotable water system piping, or natural aquatic systems. An implant inthe body, i.e. total joint, typically has the biofilms attach to theimplant and then the bacteria colony grows. A common example of biofilmsis dental plaque. It requires physical removal with a tooth brush.Physical removal is not possible when an implant is inside the body;total joint, catheter, pacemaker, etc. Therefore the must be another wayto arrest their formation by attachment to an implant and or foreignmaterial in the body. The best way, and is the subject of the invention,is to coat the implant with material the resists the biofilms attachmentas well as destroys the biofilms and bacteria upon contact.

Donlan reported the following: “For most of the history of microbiology,microorganisms have primarily been characterized as planktonic, freelysuspended cells and described on the basis of their growthcharacteristics in nutritionally rich culture media. Rediscovery of amicrobiologic phenomenon, first described by van Leeuwenhoek, thatmicroorganisms attach to and grow universally on exposed surfaces led tostudies that revealed surface-associated microorganisms (biofilms)exhibited a distinct phenotype with respect to gene transcription andgrowth rate. These biofilm microorganisms have been shown to elicitspecific mechanisms for initial attachment to a surface, development ofa community structure and ecosystem, and detachment.” (Donlan, R MBiofilms: Microbial Life on Surfaces. Emerging Infectious Diseases. Vol8 (9) September 2002).

Characklis et al. noted that the extent of microbial colonizationappears to increase as the surface roughness increases. This is becauseshear forces are diminished, and surface area is higher on roughersurfaces. Rough surfaces are common to total joint implants for thepurpose of fixation for bony ingrowth. Therefore there is compellingneed for method to coat a total joint implant.

(Characklis W G, McFeters G A, Marshall K C. Physiological ecology inbiofilm systems. In: Characklis W G, Marshall K C, editors. Biofilms.New York: John Wiley & Sons; 1990. p. 341-94).

Donlan reported that biofilms are highly resistant to most antimicrobialagents and disinfectants. (Donlan R M. Role of biofilms in antimicrobialresistance. ASAIO J. 2000; 46:S47-52). Resistant organisms such asmethicillin-resistant Staphylococcus aureus (MRSA) have also been shownto form biofilms. Therefore there is a need for biofilms destroyingreagent for MRSA including, but beyond the application to catheters.(Murga R, McAllister S, Miller J M, Tenover F, Bell M, Donlan R M.Effect of vancomycin treatment of methicillin-resistant S. aureus (MRSA)biofilms on central venous catheters in a model system. Poster No. C276presented at the 2001 American Society for Microbiology Annual Meeting,Orlando, Fla., May 23, 2001).

SUMMARY OF THE INVENTION

In its various embodiments, the present invention provides generally forcompositions and methods for reducing or preventing microbial growth,which are particularly useful for treating and promoting the healing ofwounds. Specifically, the present invention provides methods andpharmaceutical and nutraceutical compositions that reduce orsubstantially eliminate potentially pathologic microbes.

In one embodiment, a method of promoting healing of a wound in a mammalis provided; comprising administering an anthocyanin or an anthocyanidinto the mammal in need of such treatment a therapeutically effectiveamount of the anthocyanin or anthocyanidin composition or compoundwherein microbial growth is reduced and local growth hormone activity isoptimized.

In a further embodiment, a method of promoting healing of a wound in amammal is provided; comprising administering an anthocyanin metaboliteor an anthocyanidin metabolite to the mammal in need of such treatment atherapeutically effective amount of the metabolite or anthocyanidinmetabolite compositions wherein microbial growth is reduced and localgrowth hormone activity is optimized.

Further, the invention provides methods and compositions thateffectively deliver the compositions to the affected wound. The presentinvention provides compositions and methods for use in the treatment ofa variety of wound problems, including but not limited to, burns,pressure wounds, abrasions, diabetic wounds, peri-surgical and skininfections. The present invention provides compositions and methods foruse in the treatment of a variety of infections, including those fromblood transfusions. Preferably, the therapeutic compositions andcompounds are administered orally or topically; however, the therapeuticcompositions and compounds may be administered by any conventional routeincluding, for example, oral, topical, buccal, injection, pulmonary,intravenous, inhalant, subcutaneous, sublingual, or transdermal. Thoseof skill in the art can readily determine the various parameters andconditions for producing these compositions or formulations withoutresort to undue experimentation.

In one aspect, the present invention provides a pharmaceuticalcomposition for promoting wound healing, comprising: a) an anthocyanin;or b) anthocyanidin. By way of example, the anthocyanin can be selectedfrom cyanidin-3-glucoside or delphinidin-3-glucoside,cyanidin-3-galactoside, and pelargonidin-3-galactoside. Also by way ofexample, the anthocyanidins can be selected from cyanidin, delphinidin,pelargonidin, malvidin and petunidin.

In one aspect, for example, the present disclosure provides apharmaceutical composition comprising: a) an anthocyanin metabolite; orb) an anthocyanidin metabolite. In another aspect, the present inventionprovides for a pharmaceutical composition for promoting wound healing,comprising: a) an anthocyanin metabolite; or b) and anthocyanidinmetabolite. By way of example, metabolites can be selected fromprotocatechuic acid and 2, 3, 4 trihydroxybenzaldehyde. In yet anotheraspect, the present disclosure provides a pharmaceutical compositioncomprising: a) a metabolite of an anthocyanin metabolite; or b) ametabolite of an anthocyanidin metabolite. By way of example, themetabolites are metabolites selected from protocatechuic acid, and 2, 3,4 trihydroxybenzaldehyde.

Further by way of example, the pharmaceutical composition can comprise:a) an anthocyanin; or b) an anthocyanidin; and c) a pharmaceuticallyacceptable carrier. Further by way of example, the pharmaceuticalcomposition can comprise: a) an anthocyanin metabolite; or b) ananthocyanidin metabolite; and c) a pharmaceutically acceptable carrier.By way of example, the pharmaceutically acceptable carrier can beselected from, but not limited to, any carrier, diluent or excipientcompatible with the other ingredients of the composition.

Further by way of example, the pharmaceutical composition can comprise:a) an anthocyanin; and/or b) an anthocyanidin; and/or c) apharmaceutically acceptable carrier; and/or d) an acceptable deliverycarrier. By way of example, the delivery carrier can be formulated andadministered as known in the art, e.g., for topical, oral, buccal,injection, intravenous, inhalant, subcutaneous, sublingual ortransdermal Further, said topical delivery carrier may be formulated andadministered to any surface, including but not limited to skin, bone,synovium, cartilage, and implants. By way of example, the acceptabledelivery carrier can be selected from any dermal or transdermal carriercompatible with the other ingredients of the composition. In someembodiments, the acceptable delivery carrier is a biodegradablemicrosphere or a slow release bioadsorbable material. By way of example,the acceptable delivery carrier can be selected from 50/50 D, Llactide/glycolide or 85/15 D, L lactide/glycolide, both of which areamorphous physically and, therefore, are non-reactive when used as acarrier in a composition that is delivered in or to the body.

In yet another aspect and in other embodiments, the anthocyanidinprovided in any recited composition is provided less than 200 mM and inother embodiments about 100 mM. In some embodiments, the anthocyanin oranthocyanidin provided in any recited composition or method of use isprovided in a range of between 10 to 200 mM. In other embodiments, theanthocyanin metabolite or anthocyanidin metabolite provided in anyrecited composition or method of use is provided in a range of between20 to 200 mM. In yet other embodiments, the anthocyanin, anthocyanidin,or metabolites thereof provided in any recited composition or method ofuse is provided in a range of between 20 to 100 mM. In yet otherembodiments, the anthocyanin, anthocyanidin, or metabolites thereofprovided in any recited composition or method of use is provided in arange of between 20 to 50 mM. In some embodiments, the metabolite isprovided in any recited composition less than 100 mM and in others at 25mM.

In one aspect, for example, the present disclosure provides for anantimicrobial composition comprising: a) an anthocyanin metabolite; orb) an anthocyanidin metabolite. By way of example, metabolites can beselected from protocatechuic acid, 2, 3, 4 trihydroxybenzaldehyde. Byway of example, the anthocyanin, anthocyanidin and metabolites thereofcan have broad spectrum activity against disease-causing microbes. Inanother aspect of the present invention a method of treating a wound ofan individual is provided, comprising administering any of the recitedpharmaceutical compositions by topical application, transdermal, buccal,oral, gavage, and injection or intravenous.

The present disclosure also provides for methods for reducingmicroorganisms on any surface or liquid, comprising contacting a surfaceor liquid with any of the recited compositions.

The present disclosure also provides for compositions and methods forreducing microorganisms in foods comprising contacting a food with anyof the recited compositions.

The invention also provides a method of killing methicillin resistantStaphylococcus aureus (MRSA) and Pseudomonas aeruginosa biofilm coloniescomprising spraying a solution of up to 30% PCA in 70% isopropyl alcoholon a surface containing a biofilm. The surface is a solid surface,porous or semi-porous, or a cloth-like surface.

Also provided a bandage or wound dressing having a solution of PCA or 2,4, 6 trihydroxybenzaldehyde (246 THBA) or mixtures thereof impregnatedthereon, and methods of preparing same.

The invention provides a method of treating a solid surface, porous orsemi-porous, or a cloth-like surface to sanitize, sterilize, reducebacterial growth or inhibit growth of microorganisms, the methodcomprising treating the solid surface with a solution of PCA or 2, 4, 6trihydroxybenzaldehyde (246 THBA) or mixtures thereof, or in the case ofporous or semi-porous surface or cloth-like surface, treating with asolution of PCA or PCA crystals.

Included is a method of treating a medical device or surgical implant tosanitize, sterilize, reduce bacterial growth or inhibit growth ofmicroorganisms, the method comprising treating the medical device orsurgical implant with a solution of PCA or 2, 4, 6trihydroxybenzaldehyde (246 THBA) or a mixture thereof.

An embodiment of the invention includes a method of treating skin of amammal to kill microorganisms on the skin, the method comprisingtreating the skin with a solution of PCA or 2, 4, 6trihydroxybenzaldehyde (246 THBA) or a mixture thereof to killmicroorganism on the skin. The skin can be treated before a surgicalprocedure or after a surgical procedure.

Another embodiment of the invention provides a method of killing P.acnes on the skin of a patient, the method comprising applying asolution of PCA or 2, 4, 6 trihydroxybenzaldehyde (246 THBA) or amixture thereof to the skin of the patient to kill the P. acnes.

The invention also includes a solution comprising 20-30% PCA, andessential oils; as peppermint or lemon oil etc., propylene glycol andisopropyl alcohol. In addition, because liposomes have skin penetrationproperties they may be used in dermal applications with the compositionsof the invention.

The present invention also provides a covering or bandage foradministering antimicrobial compositions to a wound, wherein theantimicrobial compositions is an element of the covering or bandage.Methods for promoting healing of a wound and more specifically tomethods and compositions including the application of an anthocyanin oran anthocyanidin or metabolites thereof in the form of a covering orbandage for promoting wound healing by reducing or preventing microbialgrowth and inducing the activation of growth hormones are described.Pharmaceutical compositions containing anthocyanin or anthocyanidins ormetabolites thereof suitable for transdermal administration to a mammalfor promoting or inducing wound healing are also described.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 provides the minimum, maximum and optimum pH for growth ofmicroorganisms. Acidic environments retard proliferation of variousbacteria. Anthocyanins, anthocyanidins and main metabolites are unstablerelative to basic pH; thus, anthocyanins, anthocyanidins and mainmetabolites thereof have the potential to lower the pH of wound tissueas well as any surfaces and act as bactericidal or bacteriostatic.

FIG. 2 is the metabolic pathway of cyanidin-3-glucoside (C3G) andincludes the chemical structures of cyanidin-3-glucoside and cyanidinand their metabolites.

FIG. 3 is the chemical structure of Protocatechuic acid (PCA), adihydroxybenzoic acid, a type of phenolic acid. It is a major metaboliteof antioxidant polyphenols found in certain plants, including green tea.

FIG. 4 compares concentrations of C3G and PCA to determine optimaleffective concentrations. Bacterial burdens for P. aeruginosa werecompared after treatment with C3G or PCA at 48 and 96 hours. Aconcentration of PCA 25 mM was effective to reduce the bioburden withstatistical significance at 48 hours. C3G at 100 and 200 mMconcentrations were effective at reducing the bioburden at 48 and 96hours.

FIG. 5 is a chart disclosing potential sources of PCA.

FIG. 6 is a table providing a summary of the effectiveness of certainanthocyanins, anthocyanidins and a metabolite, including bactericidal orbacteriostatic activity. During this test, the purity, concentrationsand molecular weight of these test substances (compounds) were known.The carrier was water and the dose was accurately calculated.Delphinidin limited growth against C. perfringens, S. aureus, and MRSA.Pelargonidin limited growth of P. acnes, C. perfingens, S. aureus, MRSA,and S. pyogenes. Cyanidin CI was effective against C. difficile, Cprefringens, S. aureus ATCH 6538, S. aureus (MRSA) ATCH 33591, S.mutans, and S. pyogenes. A proprietary formulation ofcyanidin-3-glucoside (approximately 28% C3G by weight) had limitedeffectiveness during this study (18-24 hours for aerobes; 48 hours foranaerobes (C. albacans and L. casei); however, this C3G formulation, waseffective against P. acnes, E. coli, MRSA, K. pneumoniae and P.aeruginosa. Protocathechuic acid (PCA), the main metabolite fromanthocyanins and anthocyanidins, was effective against all bacteriatested as well as C. albicans and K. pneumonia. Importantly for skinwound treatment, PCA was effective against S. aureus 6538 and 33591(MRSA) and P. aeruginosa. PCA was also effective on C. albicans, whichis important considering its ability to form biofilms and difficulty intreating C. albicans when existing with a catheter or implant.

FIG. 7 is a table summarizing in vitro test results of 2, 4, 6Trihydroxybenzaldehyde and demonstrating its ability to act as anantimicrobial, including as a bactericidal or bacteriostatic.Specifically, 2, 4, 6 Trihydroxybenzaldehyde was effective against E.coli, K. pneumonia, P. aeruginosa, S. aureus 6538 and 33591 (MRSA);further it was effective against a fungi, Aureobasidium pullulans, ATCC15233.

FIG. 8A is a photographic image illustrating in vitro test results of 2,4, 6 Trihydroxybenzaldehyde against P. aeruginosa. FIG. 8B is aphotographic image illustrating in vitro test results of 2, 4, 6Trihydroxybenzaldehyde against S. aureus 33591 (MRSA). FIG. 8C is aphotographic image illustrating in vitro test results of 2, 4, 6Trihydroxybenzaldehyde against P. acnes.

FIG. 9A shows the results of a rodent back skin tape study whereconcentrations of PCA and C3G in a vehicle of water were utilized todetermine effectiveness against P. aeruginosa skin infections. FIG. 9Bshows the results of rodent back skin tape study where concentrations ofPCA and C3G in a vehicle of water were utilized to determineeffectiveness against P. aeruginosa skin infections.

FIG. 10A shows the results of an additional rodent back skin tape studyto determine effective dosages of PCA and C3G in a vehicle of water thatwould be bactericidal for P. aeruginosa. FIG. 10B shows the results ofan additional rodent back skin tape study to determine effective dosagesof PCA and C3G in a vehicle of water that would be bactericidal for P.aeruginosa. FIG. 10C shows the results of an additional rodent back skintape study to determine effective dosages of PCA and C3G in a vehicle ofwater that would be bactericidal for P. aeruginosa.

FIG. 11A shows the results of a rodent back skin study to determine theeffects of PCA and C3G on the local growth hormones in untreated skinwounds of rodents. A concentration of 25 mM PCA increased local growthhormone levels of IGF-1 at the site of the untreated skin wound. FIG.11B shows the results of a rodent back skin study to determine theeffects of PCA and C3G on the local growth hormones in untreated skinwounds of rodents. A concentration of 25 mM PCA increased local growthhormone levels of TGF-Beta at the site of the untreated skin wound. FIG.11C shows the results of a rodent back skin study to determine theeffects of PCA and C3G on the local growth hormones in untreated skinwounds of rodents. A concentration of 25 mM PCA increased local growthhormone levels of EGF at the site of the untreated skin wound.

FIG. 12A is a photographic image of rodents treated with a topicalsolution of C3G (28%); at an acidic pH, this solution maintains a purpleor red color and quickly metabolized at elevated pH levels, the C3Gchanges to a pink or even clear color. In mouse model experiments,however, as observed in the image, the purple color of C3G remained onthe rodent wound surface scar, thus indicating the pH remained acidic onthe wound surface. The C3G material on the surface was confirmed bysubsequent histology. FIG. 12B is a photographic image of tissue from astudy utilizing the homogenized wound tissue method used in this study,whereby the purple color indicates that the wound probably retained anacidic pH.

FIG. 13A shows the IGF-1 response to 25 mM PCA in various environments,including tape stripped, tape stripped and infected with P. aeruginosa,tape stripped and treated with PCA, and tape stripped infected with P.aeruginosa, and PCA treated. FIG. 13B shows the TGF-β response to 25 mMPCA in various environments, including tape stripped, tape stripped andinfected with P. aeruginosa, tape stripped and treated with PCA, andtape stripped infected with P. aeruginosa, and PCA treated. FIG. 13Cshows the EGF response to 25 mM PCA in various environments, includingtape stripped, tape stripped and infected with P. aeruginosa, tapestripped and treated with PCA, and tape stripped infected with P.aeruginosa, and PCA treated.

FIG. 14 is a photographic image of a cross section of rodent skin.

FIG. 15 is a photographic image of a cross section of rodent skin.

FIG. 16 is a photographic image of a cross section of rodent skin.

FIG. 17 is a photographic image of a cross section of rodent skin.

FIG. 18 is a photographic image of a cross section of rodent skin.

FIG. 19 is a photographic image of a cross section of rodent skin.

FIG. 20 is a photographic image of a cross section of rodent skin.

FIG. 21 is a photographic image of a cross section of rodent skin.

FIG. 22 is a photographic image of a cross section of rodent skin.

FIG. 23 is a photographic image of a cross section of rodent skin.

FIG. 24 is a photographic image of a cross section of rodent skin.

FIG. 25 is a photographic image of a cross section of rodent skin.

FIG. 26 is a photographic image of a cross section of rodent skin.

FIG. 27 is a photographic image of a cross section of rodent skin.

FIG. 28 is a photographic image of a cross section of rodent skin.

FIG. 29 is a photographic image of a cross section of rodent skin.

FIG. 30 is a photographic image of a cross section of rodent skin.

FIG. 31 is a photographic image of a cross section of rodent skin.

FIG. 32 provides a chart studying the effectiveness of anthocyanin andanthocyanidin metabolites against various microbes, including P. acnes,C. difficile, E. coli 8739 and 43895, S. Aureus 6538, 33591, P.Aeruginosa 9027, MRSA 51625 and Legionella 43662, methicillin resistantstaphylococcus epidermis (MRSE), including MRSE ATCC 51625, and others.

FIG. 33 provides a chart summarize results of testing PCA againstPseudomonas biofilm.

FIG. 34 provides a chart summarize results of testing PCA againstPseudomonas biofilm.

FIG. 35 provides a chart summarize results of testing PCA against MRSAbiofilm.

FIG. 36 provides a chart summarize results of testing PCA against MRSAbiofilm.

FIG. 37 provides a chart summarize results of testing PCA againstPseudomonas biofilm.

FIG. 38 provides a chart summarize results of testing PCA against MRSAbiofilm.

FIG. 39 provides a chart of results of testing PCA against P. acnes onthe skin.

FIG. 40 shows the results for a single spray of 30% PCA in isopropylalcohol on 10 million biofilms colonies of Pseudomonas aeruginosa.

FIG. 41 shows the concentration of 30% has lesser effect on MRSA, butstill 90%.

FIG. 42 provides a composite of the results based only for no growthcultures following treatment by each solution. *Note that the 1% PCA wasnot included.

FIG. 43 provides the summation percentages of “no growth.”

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise indicated, all technical and scientific terms usedherein shall have the same meaning as is commonly understood by one ofordinary skill in the art to which the disclosed subject matter belongs.Unless otherwise indicated, the following definitions are applicable tothis disclosure. All publications referred to throughout the disclosureare incorporated by reference in their entirety. To the extent anydefinition or usage provided by any document incorporated herein byreference conflicts with the definition or usage provided herein, thedefinition or usage provided herein controls.

As used in the specification and claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a composition” includesmixtures or combinations of two or more such compositions.

Throughout the specification and claims, the word “comprise” andvariations of the word, such as “comprising” and “comprises,” means“including but not limited to,” and is not intended to exclude, forexample, other components, extracts, additives, or steps. It is alsocontemplated that embodiments described as “comprising” components, theinvention also includes those same inventions as embodiments “consistingof” or “consisting essentially of.”

Ranges can be expressed herein as “approximately” or from “about” oneparticular value, and/or to “about” another particular value. When sucha range is expressed, another embodiment includes from the oneparticular value and/or to the other particular value.

A weight percent of a reagent, component, or compound unlessspecifically stated to the contrary, is based on the total weight of thereagent, component, composition or formulation in which the reagent,component, or compound is included, according to its usual definition.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant decrease or lower a characteristic (e.g.,inflammation, growth or viability of microorganisms).

By “promote” or other forms of the word, such as “promoting,” is meantto induce a particular event or characteristic, or delay the developmentor progression of a particular event or characteristic, or to minimizethe chances that a particular event or characteristic will occur.

“Treat” or other forms of the word, such as “treating,” “treatment” ortreated,” is used here to mean to administer a composition or to performa method in order to induce, reduce, eliminate, and prevent acharacteristic (e.g., inflammation, growth or viability of microbes). Itis generally understood that treating involves providing an effectiveamount of the composition to the mammal or surface for treatment.

The term “vehicle” or “vehicle carrier” as used herein refers to meanthe manner in which the reagents or compositions may be delivered,including as a liquid, salve, soap, foam, cream, solution, gel, spray,powder, wipes, antibacterial treatments, wipes and the like.

The term “wound” or “wound associated condition” as used herein refersto a medical condition when the integrity of any tissue is compromised(e.g., burns, skin breaks, bone breaks, muscle tears, tendon injuriespunctures, surgical incision sites, microdermabrasion site, skin graftsite,). A wound may be caused by any act, infectious disease, underlyingcondition, fall, or surgical procedure. A wound may be chronic, such asskin ulcers caused by diabetes mellitus, or acute, such as a cut orpuncture from a sharp object, an animal bite or a gunshot.

The term “growth factors” or “local growth factors” include but are notlimited to, fibroblast growth factor (FGF), FGF-1, FGF-2, FGF-4,platelet-derived growth factor (PDGF), insulin-binding growth factor(IGF), IGF-1, IGF-2, epidermal growth factor (EGF), transforming growthfactor (TGF), TGF-β, TGF-α, and collagen growth factors, and/orbiologically active derivatives of these growth factors.

By “bactericidal” or “antimicrobial” is meant the ability to effect(e.g., eliminate, inhibit decrease, or prevent) microorganism growth,viability, and/or survival at any concentration. It also means kill themicroorganism.

By “bacteriostatic” is meant the ability to effect (e.g., stabilize orprevent future growth or prevent new growth) microorganism growth at anyconcentration. A bacteriostatic compound, agent or reagent does noteliminate or kill the bacteria.

By “additive” or “food additive” is meant to the use as a component ofany food (including any substance intended to use in producingmanufacturing, packing, processing, preparing, treating, packaging,transporting, or holding food).

By “antiseptic” is meant an antimicrobial reagent or composition that isapplied to any surface, including skin or tissue, to effect (e.g.,eliminate, inhibit, decrease or prevent) microorganism growth,viability, and/or survival.

By “disinfect” or other forms of the word, such as “disinfectant” or“disinfecting,” is meant decrease or lower a characteristic (e.g.,eliminate, reduce, inhibit, decrease, or prevent) microorganism growth,viability or survival at any concentration. It is generally understoodthat disinfect involves providing an effective amount of the compositionto any surface, but particularly solid surfaces, whether smooth orporous or semi-porous, or cloth-like surfaces.

By “sanitize” or other forms of the word, such as “sanitizer” or“sanitizing,” is meant decrease or lower a characteristic (e.g.,eliminate, reduce, inhibit, decrease, or prevent) microorganism growth,viability or survival at any concentration. It is generally understoodthat sanitizing involves providing an effective amount of thecomposition to any surface. Further, it is generally understood thatsanitizing solutions and sanitizing components are those solutions thatmay be safely used on food-processing equipment and utensils and onother food-contacting conditions.

By “sterilize” it is meant to kill microbes on the article beingsterilized. Sterilize and sterilization include cold sterilizationmethods.

By “isolated” or “an isolate” as it refers to either the compounds orreagents described herein means not 100% by weight but ratherapproximately 95% to 97% of the compound or reagent by weight.

The term “alkyl” as used herein is a branched or unbranched hydrocarbongroup of 1 to 20 carbon atoms. Non limiting examples include methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl,eicosyl, tetracosyl, and the like. Further, the alkyl group can also besubstituted or unsubstituted.

The term “alkoxy” or “alkyoxy group” as used herein refers to a branchedor unbranched hydrocarbon chain having from 1 to 15 carbons and linkedto oxygens. Non-limiting examples include methoxy, ethoxy and the like.

The term “ProC3G™” (commercially available ChromaDex®, Inc. Irvine,Calif. product) means a cyanidin 3-glucoside anthocyanin extracted fromblack rice and containing approximately 28% cyanidin 3-glucoside byweight with an additional 5% other anthocyanins.

The term “medicament” as used herein refers to any wound treatment,including but not limited to the group consisting of burn reliefmedications, anesthetic agents, wound cleansers, antiseptic agents, scarreducing agents, immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,acne treating agents, sunscreen agents, dermatological agents,antihistamine agents, antibacterial agents, bioadhesive agents,inhibitors of prostaglandin synthesis, antioxidants, and mixturesthereof.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer.

The term “nutraceutical” as used herein refers to any food stuff,including a dietary supplement or fortified food, provided for potentialhealth and medical benefits.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, and methods, examples ofwhich are illustrated in the following description and examples, and inthe figures and their descriptions.

The present invention provides methods, compositions and uses fortreating and promoting healing of a wound. More specifically, themethods and compositions described herein include the administration ofan anthocyanin or an anthocyanidin or metabolites thereof, preferablyPCA and 2,4,6 Trihydroxybenzaldehyde (2,4,6 THBA) for promoting woundhealing by reducing or preventing microbial growth and inducing theactivation and or optimization of growth hormones.

The methods and compositions are used for the treatment of mammals,including humans. As with humans, there is a need for new antimicrobialcompositions for the treatment of animals, including equine, canine andfeline, due to resistance or allergic reactions to current antimicrobialcompositions or agents. Therefore, the methods and compositionsdisclosed herein will be useful for the treatment and promotion of woundhealing in livestock as well as domestic pets and will havebroad-spectrum activity against microbes.

The present invention provides a broad spectrum antibiotic. The inventorhas shown the PCA and 2,4,6 Trihydroxybenzaldehyde (2,4,6 THBA) are abroad spectrum antibiotic. The inventor has shown that the parentanthocyanins or an anthocyanidins had limited antibiotic propertiesincluding cyanidin chloride. Further the subsequent acids in themetabolic chain; vanillic and hippuric acid had little activity as well.The inventor has thus identified the specific metabolites in the chainthat had the antibiotic properties.

Methods and compositions described herein include the administration ofan anthocyanin or an anthocyanidin or metabolites thereof, preferablyPCA and 2,4,6 Trihydroxybenzaldehyde (2,4,6 THBA) as a skin sterilizer(antiseptic). The normal human skin bacterial resident flora is onesource of surgical site infections. There are several available topicalapplication means of decreasing the bacterial presence prior to surgery.Two such commercially available preparations are Chloroprep® andBetadine®. Reports of their effectiveness in the medical literature showresidual bacteria. Propionibacterium acnes is one such organism.

Therefore there is a need for a more effective means of pre-operativehuman skin treatment. Protocatechuic acid (PCA), a phytochemicalmetabolite, may be one such chemical reagent. We have shown its broadspectrum anti biotic properties in vitro. In addition, in vivo testingin animals has demonstrated its bactericidal properties on tapedstripped open skin wounds. Therefore it was reasonable to study itspotential application as a skin antiseptic. For example, a compositionof PCA and 2,4,6 Trihydroxybenzaldehyde (246 THBA) or mixtures thereofcan be applied to the skin or wound in advance or after a surgical ordental procedure to kill bacterial present on the skin or wound. It hasbeen shown (See FIGS. 6 and 32) that PCA and 2,4, 6 THBA have theability to kill a wide spectrum of microbes. For example, FIG. 6provides the results of testing showing that protocatechuic acid (PCA),the main metabolite from anthocyanins and anthocyanidins, was effectiveagainst all bacteria tested as well as C. albicans and K. pneumonia.Importantly for skin wound treatment, PCA was effective against S.aureus 6538 and 33591 (MRSA) and P. aeruginosa. PCA was also effectiveon C. albicans, which is important considering its ability to formbiofilms and difficulty in treating C. albicans when existing with acatheter or implant. In FIG. 32, it is shown that PCA is effectiveagainst C. difficile, P. acnes 6919, E. coli 8739 and 43895, S. Aureus6538, S. Aureus 33591, P. Aeruginosa 9027, methicillin resistantstaphylococcus epidermis (MRSE), including MRSE ATCC 51625, andLegionella 43662, and others. FIG. 32 shows that 2, 4, 6 THBA iseffective against E. coli 8739 and 43895, S. Aureus 6538, S. Aureus33591, P. Aeruginosa 9027, methicillin resistant staphylococcusepidermis (MRSE), including MRSE ATCC 51625, and Legionella 43662, andothers. In addition, by treating the skin, the compositions promotewound healing by reducing or preventing microbial growth and inducingthe activation and or optimization of local skin growth hormones.

The present invention also provides methods, compositions and uses fortreating surfaces (solid, smooth, porous or semi-porous, or cloth-like)and liquids to reduce microbial growth or to sanitize or sterilize thesurface. More specifically, the methods and compositions describedherein include contacting any surface with a composition comprising ananthocyanin or an anthocyanidin or metabolites thereof (preferably PCAand 2,4,6 Trihydroxybenzaldehyde (2,4,6 THBA)) thereby reducing orpreventing microbial growth on said surface, or to sanitize or sterilizethe surface. The surfaces can be in the health care setting, sportssetting or even food preparation settings or any setting where sterilesurfaces are required.

The compositions can be applied to solid surfaces such as implants, orsolid surfaces like operating tables, benches, equipment, patient beds,etc. or surgical instruments to sanitize or sterilize the surface. Forexample, in the case of implants, the implants can be treated and coatedwith the compositions before inserted into the patient. Likewise, theinstruments may be treated before use on a patient. Solid surfaces suchas operating tables, other equipment and other surfaces can be treatedas well by spraying of the surface with compositions comprising PCA,2,4,6 THBA or mixtures thereof.

In addition, the compositions can be applied to smooth, porous orsemi-porous, or cloth-like surfaces such as wound dressings, bedding,vascular implants, bandages, etc. The material can be treated with thePCA solution and then used immediately or the material can be allowed todry and then used. For example, a bandage can be treated with PCA andthen allowed to dry and store (for about up to 2 years known shelflife). When needed, depending upon the nature of the wound, the bandagecan either be applied directly to the wound or can be wetted with water,70-90% isopropyl alcohol, saline or propylene glycol and/or essentialoils and then applied to the wound. It is preferred to use propyleneglycol and an essential oil as they enhance the absorption of PCA intothe skin.

When the antimicrobial composition of this invention is applied to as anelement of a covering or bandage, to adhere to a surface to be treated,such as a wound, the antimicrobial composition generally can include aconcentration of the PCA or 246 THBA or mixtures thereof of at least1.24% and 30% by weight of the compound depending upon the chemicalnature of the vehicle, the target being treated and the species ofbacteria to be treated. In certain embodiments a 25 mM concentration ofPCA is applied to the bandage. In Trans Pharm's independent laboratoryexperiment 367 on tape stripped wounds or rodents, the data demonstratedthat C-3-G at 100 and 200 mM significantly decreased the bacterialburden of P. aeruginosa in skin tissue at 48 and 96 hours. The treatmentwith 25 mM PCA significantly decreased the bacterial burden of P.aeruginosa at the 48 hour interval.

Further, the isolated anthocyanins, anthocyanidins, or metabolitescompounds this invention will be between 20-30% by weight of thecompound for one intended use and more preferably, between 1.24% and 30%by weight of the compound depending upon the chemical nature of thevehicle, the target being treated and the species of bacteria to betreated. Preferably the antimicrobial composition comprises PCA or 2, 4,6 Trihydroxybenzaldehyde (2,4,6 THBA) or mixtures thereof. Thesebandages can promote wound healing not only as they are effectiveagainst killing microbes, but they also promote healing by inducing theactivation and or optimization of local skin growth hormones.

Further, the methods and compositions described herein include addingthe composition comprising an anthocyanin or an anthocyanidin ormetabolites thereof to liquid or fluid, including other sanitizingsolutions and/or sanitizing components, thereby reducing or preventingmicrobial growth on said surface. Further still, the methods andcompositions described herein include adding the composition comprisingan anthocyanin or an anthocyanidin or metabolites thereof to any othervehicle, including but not limited to a powder, paste, cream foam, gel,wipes, other sanitizing components and the like thereby reducing orpreventing microbial growth on said surface.

Included in the invention are compositions for, and methods ofdestroying, killing or significantly reducing a bacterial biofilms.Biofilms are comprised of bacteria that form colonies and produce asurrounding matrix film to protect themselves. The biofilm formingbacteria can form colonies that attach to foreign bodies, each other andtissues. The bacteria aggregate in clusters and are surrounded byextracellular polymer matrix. The biofilms are hard to destroy andtherefore kill the underlying bacteria and provide the basis for much ofthe antibiotic resistance that has developed. Biofilms can be foundattached to surfaces such as implants and catheters and they also can beembedded in the biological host, such as for example cystic fibrosiswounds. MRSA biofilms play a role in many device-related infections suchas native valve endocarditis, otitis media, urinary tract infections,cystic fibrosis, acute septic arthritis, total joint implantation,catheters, pacemakers, etc. The formation of a biofilm is a two-stepprocess: 1. Adherence of cells to a foreign body surface; and 2.Accumulation of cells to form multilayered cell clusters. A trademark ofbiofilm formation in staphylococci is the production of polysaccharideintercellular adhesion.

The present invention thus provides a composition that destroysbiofilms. The composition is preferably PCA. The PCA may be mixed with70% isopropyl alcohol and or small amount of essential oil; i.e. lemon,peppermint, etc. The concentration of PCA can be anywhere from about 20%by weight PCA to 100% PCA. Preferably the concentration of PCA varieswith the intended purpose from 1.24%, 20%, 30%, 20-50% or 20-40% or20-30% by weight. When the PCA is used in a crystal form, then thecrystals can be up to 100% PCA by weight. The PCA may be in the form ofcrystals that are embedded into a material such as a cloth or a mesh,such as titanium or stainless steel. The crystals are applied to themetal where there is surface configuration that provides for housing ofthe crystal on the surface. The same is for cloth material that has meshor surface to house the physical crystals by size. The may be seen invascular grafts. They remain in place in crystal form until activatedwhen subject to fluid common to the mammalian body.

The inventor has shown that a composition comprising PCA was able tostop the formation of a biofilm as well as kill bacterial in alreadyformed biofilms. The biofilms tested were Pseudomonas Aeruginosa ATCC700888 and Staphylococcus aureus ATCC 33591 (MRSA). The tests aredescribed in more details below and in Examples 7 and 8.

It should be noted that the dose of the parent anthocyanin,anthocyanidin, and or the main metabolites, PCA and or 246 THBA varywith the application and the vehicle. For instance the dose used on anopen rodent taped stripped skin would be conditioned by the wound'stolerance of the vehicle which in this case would be water, normalsaline or like. The amount of PCA for instance that can be dissolved inwater is 1.24 grams per 100 ml. In the rodent the effective dose ofC-3-G and PCA were expressed in micromolars (mM) as 25 to 200 mM dose.Conversion factors are necessary to make comparisons to larger dosesused in other applications. 78 mM (mmole/L) PCA would be 12.021 g PCAper Liter. Therefore 25 mM/L would be 3.852 grams per liter or 0.0038mg/ml. The fluid applied was 100 mL or 0.1 milliliter. Therefore, forexample, the dose of PCA that was bactericidal on Pseudomonas aeruginosaon taped stripped rodent skin was 0.00038 grams. When treating intactnormal human skin as in the Loma Linda Medical School testing, it wasshown the 1.24 gram in 100 ml of water would only decrease the number ofcolonies of Propionibacterium acnes. Note that there was no skinpenetration properties to water by this method. The increase of PCAconcentration was possible to as much as 17% when in composition ofmatter was 70% isopropyl alcohol with propylene glycol and essences ofpeppermint oil. This composition had skin penetration properties.However the amount of liquid applied was no more than one milliliter bycotton swab. Therefore the topical dose on intact human skin was 1.7grams (estimated). This is many times that required for traumatizedtaped stripped skin.

The amount of PCA necessary for coating metal and or cloth was 20 to 30%PCA or 20-30 grams per 100 ml of 70% isopropyl alcohol. Thesecompositions allowed for higher concentrations, and also evaporatedrapidly to dry state of PCA crystals on the metal or cloth.

The next variable was the species of bacterial to be eradicated. It wasnoted that a 10% concentration of PCA was not effective on Pseudomonasaeruginosa. See FIG. 33. However a 20% concentration was effective. SeeFIG. 34.

The biofilms destroying properties of coating metal and linen forMethicillin resistant Stapylococcus aureus required higher dose thanPseudomonas aeruginosa. See FIG. 35. However at concentration of 30%, itwas effective. See FIG. 36. The application on an implant allowed to dryhad the above results. However, when a glass surface is covered with 10million biofilms colonies the results differ with the concentration andthe bacteria biofilms to be eradicated. The glass surface experimentsare described in example 8. Generally, Glass slides were inoculated atTime 0. Batch phase was performed for 6 hours to allow for biofilmformation on the glass slides. The drip flow mechanism was then turnedon to provide a continuous flow of nutrients to the glass slides over 48Hours. After 48 hours, 2 sets of glass slides were sprayed with a 30%PCA solution. One set was removed and analyzed for biofilm reductionafter 30 minutes. The other set was removed after 60 minutes. 2 sets ofcontrol slides were also removed and analyzed after 30 minutes and 60minutes. The control slides were not treated with 30% PCA and were usedfor comparative purposes. The Log Reduction Calculations were performedas follows: The mean 30% PCA treated samples were compared to the meanpositive control samples, per time point evaluated. The results were asfollows for a single spray of 30% PCA in isopropyl alcohol on 10 millionbiofilms colonies of Pseudomonas aeruginosa. See FIG. 40. Theconcentration of 30% has lesser effect on MRSA, but still 90%. See FIG.41. These experimental concentrations of biofilms covered pathogens thatfar exceed the concentrations and numbers found in practice.

The above experimental results show discovery of dose and compositionrequirements for any given intended use related to nature of the wound,the disinfectant intentions, environmental conditions of coating and orspraying plus consideration of the bacterial species and biofilms to bedestroyed.

Regarding wound healing and the treatment of wounds comprising theanthocyanins and anthocyanidins and their metabolites using compositionsof the present invention, these compositions also have broad spectrumactivity against a wide range of microbes. These compositions, however,may also be used in combination with other wound treatments, includingother antimicrobials. In certain embodiments, the additionalantimicrobial is not sulfamethoxazole.

The identification of anthocyanins and anthocyanidins or combinations ofanthocyanins, anthocyanidins or their metabolites that are bactericidalor antimicrobial was determined by conducting in vitro testing.Anthocyanidins that were tested at 100 mM (44.938 grams per liter) withless than one milliliter per dose included delphenindin, pelargonindin,and cyanidin CI and cyanidin-3-glucoside. Protocatechuic acid and 2, 4,6 trihydroxybenzaldehyde, the anthocyanidin metabolites, were alsotested at the same concentrations. Referring to FIGS. 6-8, delphinidinlimited growth against C. perfringens, S. aureus, and MRSA. Pelargonidinlimited growth of P. acnes, C. perfingens, S. aureus, MRSA, and S.pyogenes. Cyanidin CI was effective against C. difficile, C prefringens,S. aureus ATCH 6538, S. aureus (MRSA) ATCH 33591, S. mutans, and S.pyogenes. C3G (approximately 28% by weight) had limited effectivenessduring this study (18-24 hours for aerobes; 48 hours for anaerobes (C.albacans and L. casei). This proprietary C3G formulation, however, waseffective against P. acnes, E. coli, MRSA, K. pneumoniae and P.aeruginosa. Protocathechuic acid (PCA), the main metabolite fromanthocyanins and anthocyanidines, was effective against all bacteriatested as well as C. albicans and K. pneumonia. Importantly for skinwound treatment, PCA was effective against S. aureus 6538 and 33591(MRSA) and P. aeruginosa. PCA was also effective on C. albicans, whichis important considering its ability to form biofilms and difficulty intreating C. albicans when existing with a catheter or implant. 2, 4, 6Trihydroxybenzaldehyde was effective against E. coli, K. pneumonia, P.aeruginosa, S. aureus 6538 and 33591 (MRSA); it also was effectiveagainst A. pullulans, ATCC 15233, a fungi.

While specific dosages of certain anthocyanins and anthocyanidins weredetermined to have the above mentioned effects against certain bacteria,in vivo testing were conducted to determine optimal dosages and toconfirm the ability of a topical application of these compounds to haveantimicrobial effect while prompting healing of a wound. It washypothesized that certain dose and interval topical application of awater soluble solution of PCA and/or C3G (28% of C3G by weight) atcertain concentrations based upon molecular weight would kill or reducethe bioburden of Pseudomonas aeruginosa while healing the wound asevidence by optimization of the local growth hormones and confirmed byhistological evidence. Referring to FIG. 9, a decrease in bacterialburden in the skin at 96 hours days was noted (CFU means colony formingunits). A concentration of 50 mM of PCA was found to be most effective;higher concentrations of PCA were not as effective at decreasingbacterial burdens. The most effective concentration of C3G was 100 mM.Importantly, histological evaluations of skin samples from the studyconfirmed healing at 48 and 96 hours with proliferation ofparafollicular cells and migration to cover the skin surface. There wasminimal inflammation in the dermis. There was collagen proliferation inthe dermis. In this application, in some embodiments, anthocyanins oranthocyanidins and metabolites thereof are provided in concentrations ofabout 10 to 200 mM. In other embodiments, anthocyanins, oranthocyanidins, or metabolites thereof are provided in any recitedcomposition or method of use in a range of between 20 to 200 mM. In yetother embodiments, the anthocyanin, anthocyanidin, or metabolitesthereof provided in any recited composition or method of use is providedin a range of between 20 to 100 mM. In yet other embodiments, theanthocyanin, anthocyanidin, or metabolites thereof provided in anyrecited composition or method of use is provided in a range of between20 to 50 mM. In a preferred embodiment, anthocyanins or anthocyanidinsand or their metabolites are provided in concentrations of about 100 mMor less.

As provided in FIG. 1, bacteria have a range of pH at which growth isoptimized, and most bacteria are more viable at basic pH ranges.Generally, anthocyanins, anthocyanidins and their metabolites also havean acidic pH and have the potential to have bactericidal orbacteriostatic modes of action. Because C3G and PCA reagents have anacidic pH, their bactericidal or bacteriostatic mode of action is bydirect contact with the bacteria.

Anthocyanins and anthocyanidins were further studied to determineeffects on wound healing, including whether they had any effect on theoptimization of local growth hormone activity at the wound site alongwith other supporting histological evidence of promoting healing.

Local growth hormones are important substances in the control of woundhealing. Equally as important, however, is to optimize the amount ofthese hormones desirable for promoting wound healing while avoiding scarformation and keloids.

Examples of common local growth hormones related to skin wound healinginclude Epidermal growth factor (EGF), Insulin-like growth factor-1(IGF-1) and Transforming Growth Factor-Beta (TGF-β). Epidermal growthfactor or EGF is a growth factor that stimulates cell growth,proliferation, and differentiation by binding to its receptor EGFR.IGF-1 is important in skin repair by stimulating keratinocyteproliferation and migration as well as collagen production byfibroblasts. Its expression is important during wound healing such thatretarded healing has been correlated with reduced IGF-1 levels. Whilelocal administration of IGF-1 to wound sites enhanced wound closure andstimulated granulation tissue formation, increased IGF-1 receptorexpression was reported in chronic wounds and in hypertrophic scars.Additionally, IGF-1 stimulation was associated with increased invasivecapacity of keloid fibroblasts. Systemic delivery of IGF-1 also causedhyperglycemia, electrolyte imbalance, and edema. Therefore it isdesirable to have slightly elevated but not over elevated IGF-1 by atreatment modality. TGF-β also is important in skin would healing;however, it is considered a pro-fibrotic growth factor and increasedlevels of TGF-β or prolonged presence has been identified as causinghypertrophic scaring.

Referring to FIGS. 11-13, tests were performed on rodent skin to explorethe effects of PCA on the local growth hormones in rodent skin. Aconcentration of 25 mM PCA increased local growth hormone levels at thesite of the untreated skin wound. In particular, FIG. 11 demonstratesthat a single reagent or compound would optimize local growth hormonesto promote healing without scarring. Approximately 25 mM PCA was theoptimal reagent and dose. As demonstrated in FIG. 13, optimization ispossible using the compositions of the present invention. In FIG. 13,all three local growth hormones were lowered in the simulated clinicalpathological environment (stripped and infected); however, the loweringof these hormones was not to the extent of absences. Hence, thenecessary IGF-1 is still above the controls in this environment;however, the scar forming properties of the other two hormones have beenmarkedly reduced. Therefore, optimization of local growth hormones isachieved. In FIG. 13, the optional concentration of PCA was confirmed as25 mM PCA in this situation and environment, meaning local growthhormone growth levels were optimized at this dosage such that IGF-1 asmoderately elevated while TGF-β and EGF levels were decreased. This isimportant to promote wound healing while preventing potential scarring.

The therapeutic effective dose may vary depending on a wide variety offactors. For instance, the dose may vary depending on the formulation,method of application of the therapeutic reagent or combination withother reagents, or compositions, compounds or combination ofcompositions or compounds to the wound.

Methods (General)

According to one aspect of this invention, there is provided anantimicrobial composition and a method of promoting wound healing byreducing microbial growth. A method of promoting healing of a wound in amammal is provided, comprising administering an anthocyanin or ananthocyanidin to the mammal in need of such treatment a therapeuticallyeffective amount of the anthocyanin or anthocyanidin compound whereinmicrobial growth is prevented or reduced and local growth hormoneactivity is optimized.

In a further embodiment, a method of promoting healing of a wound in amammal is provided; comprising administering an anthocyanin metaboliteor an anthocyanidin metabolite to the mammal in need of such treatment atherapeutically effective amount of the metabolite or an anthocyanidinmetabolite compound wherein microbial growth is reduced and local growthhormone activity is optimized.

In some embodiments, a method of promoting healing a wound furthercomprises applying a wound dressing or bandage that has been treatedwith a composition of PCA or 246 THBA or mixtures thereof.

In yet another aspect of the present invention, a method of treating P.acnes in a patient in need thereof is provided, comprising administeringan anthocyanin metabolite to the patient in need of treatment in antherapeutically effective amount of protocatechuic acid wherein growthof P. acnes is reduced. FIG. 32 shows that both C-3-G and PCA were ableto kill P. acnes, specifically P. acnes 6919.

In another aspect of the present invention, a method of prophylacticallytreating a preoperative skin incision site is provided, comprisingadministering an anthocyanin, an anthocyanidin and/or a metabolite to apatient in need of such treatment an effective amount of the anthocyaninor anthocyanidin compound wherein microbial growth is prevented orreduced.

In another aspect of the present invention, a method of disinfecting asurface comprising contacting said surface with an anthocyanin, ananthocyanidin and/or a metabolite thereof in an effective amount of theanthocyanin, anthocyanidin and/or metabolite compound wherein microbialgrowth is prevented, reduced or eliminated and, further, where themicrobial growth that is reduced is methicillin resistant Staphylococcusaureus (MRSA).

In another aspect of the present invention, a method of disinfecting orsterilizing a surface is provided comprising contacting said surfacewith an anthocyanin, an anthocyanidin and/or a metabolite thereof orcombination thereof in an effective amount of the anthocyanin,anthocyanidin and/or metabolite or combination thereof wherein microbialgrowth is prevented, reduced or eliminated and, further, where themicrobial growth that is reduced may be an endogenous or exogenoussource, including but not limited to P. acnes, S. aureus, P. aeruginosa,E. coli, S. epidermidis, S. pneumonia, Streptococcus species, C.difficile and Legionella.

In another aspect of the present invention, a method of post-operativetreating a post-operative skin site is provided, comprisingadministering an anthocyanin, an anthocyanidin and/or a metabolite to apost-operative skin site, such as a skin graft, skin graft donor site, amicrodermabrasion site, or a surgical incision site, in an effectiveamount of the anthocyanin, anthocyanidin and/or metabolite compoundwherein microbial growth is prevented, reduced or eliminated and localgrowth hormone production is optimized.

This disclosure also provides for a method comprising contacting asurface with an effective amount of the antimicrobial composition. Bythe term “effective amount” of a composition as provided herein is meantan amount of a composition sufficient to provide the desired benefit,either bactericidal or bacteriostatic (e.g., reduction or prevention ofmicroorganism growth or survival). As disclosed herein, the exact amountrequired will vary from use to use depending on a variety of processingparameters, as understood by one of ordinary skill, such as the type ofsurface, the type of microorganism to be treated, the surface size, themode of deliver (e.g., aerosol, spraying or dipping), and the like.Determination of what constitutes an “effective amount” is made byroutine testing with known concentrations and adjusting thoseconcentrations as needed to obtain the desired benefit and can bedetermined by one of ordinary skill in the art using only routineexperimentation.

When the antimicrobial composition of this invention is applied to asurface to be treated, the antimicrobial composition generally caninclude a concentration of the anthocyanins and anthocyanidins of atleast 25 mM concentration, not including the carrier. Further, theisolated anthocyanins, anthocyanidins, or metabolites compounds thisinvention will be between 90%-97% by weight of the compound, and morepreferably, between 95%-98% by weight of the compound.

When the antimicrobial composition or compositions of this invention areapplied to a surface to be treated may be diluted for use as a sanitizeror as a preventive or prophylactically, and at greater concentrationsfor treatment.

In another aspect of the invention provides a method of inhibitinggrowth of a biofilm on a solid, smooth, porous or semi-porous, orcloth-like surface (such as but not limited to a cloth, wound dressing,bandage, heart or vessel grafts) by treating the surface with acomposition of the present invention, preferably comprising PCA. Thesurface can be any solid, smooth, porous or semi-porous, or cloth-likesurface, including implants that are inserted into a patient.

Compositions

Disclosed herein, in one aspect, are antimicrobial compositions. Thedisclosed antimicrobial reagents and compositions can be used toeliminate, reduce, and/or prevent microorganism growth, viability, orsurvival.

The present invention thus provides a composition that destroys orinhibits growth of a biofilm. The composition is preferably PCA. The PCAmay be mixed with 70% isopropyl alcohol. The concentration of PCA can beanywhere from about 20% PCA to 100% PCA. Preferably the concentration ofPCA is about 20-50% by weight, or is about 20-40% by weight or is about20-30% by weight or is 30% to 50% by weight. Additionally, the PCA maybe in the form of crystals that are embedded into a material such as acloth or a mesh, such as titanium or stainless steel.

For compositions applied to the skin or a bandage the PCA crystals needto be dissolved in a liquid. The inventor found that PCA in water onlydecreased the colonies of P. acnes in and on human skin as the PCAconcentration he was able to achieve when dissolving in water waslimited to 1.24%. The inventor had to increase the concentration of PCAabove 1.24% and to do this, the inventor discovered that he would haveto use isopropyl alcohol instead of water to dissolve the crystals. Theuse of isopropyl alcohol in the combination was not an obvious choicebecause isopropyl alcohol alone only is marginally effective in killingbacteria. So even after using isopropyl alcohol to dissolve the PCA, theinventor discovered that even at 10% PCA in isopropyl alcohol, thecomposition was only marginally effective. So the inventor discoveredthat he had to increase the PCA to at least 17% in isopropyl alcoholplus add two skin penetration vehicles (propylene glycol and essentialoil) to achieve the desired and most effective means of controlling P.acnes, which are deep in the skin. Accordingly, the invention provides acomposition comprising about 17 to 40%, or 17 to 30% or 17 to 20% byweight of PCA, isopropyl alcohol, propylene glycol and an essential oil,preferably of peppermint, or a citrus fruit (i.e. lemon, grapefruit,orange, lime, etc.). This composition is useful in the methods describedin the invention, for example as a skin antiseptic as a surfacedisinfectant, as a spray to disinfect a surface, etc. Further, theinventor found that neither propylene glycol or essential oil alone orin combination absent the isopropyl alcohol provide enough concentrationof PCA to be above the effective range of 10% concentration. Thereforethe composition of the invention, to be effective should have at leastPCA at 17+% by weight in at least 70-90% isopropyl alcohol, propyleneglycol (15 mls in a 105 ml total solution) and the essential oil; i.e.peppermint or lemon etc.

The invention further provides a composition of PCA wherein thecomposition comprises or consists of PCA that can be applied directly orprovided in various vehicles depending upon the application. PCA of 1.24grams in 100 milliliters of water was effective on open wound. Acomposition of 70% isopropyl alcohol, propylene glycol and essentialpeppermint oil was also effective in use as a skin antiseptic,especially against P. acnes. Studies showed that higher concentration of10% PCA (20 grams in 90 milliliters of 70% isopropyl alcohol) was moreeffective than PCA in water. However the following concentration wasmore effect than just PCA in isopropyl alcohol—the composition consistedof PCA (20 grams) 70% isopropyl alcohol (85 ML), propylene glycol (15ml) and an essential oil (5 ml).

The invention also provides bandages, wound dressings and the likecomprising PCA or 246 THBA.

In some embodiments and aspects, the disclosed antimicrobial compositioncan be selected from the list of anthocyanins, anthocyanidins,metabolites of anthocyanin and anthocyanidin metabolites, or acombination thereof. By way of example, the anthocyanin can be selectedfrom cyanidin-3-glucoside or delphinidin-3-glucoside,cyanidin-3-galactoside, and pelargonidin-3-galactoside. Also by way ofexample, the anthocyanidins can be selected from cyanidin, delphinidin,pelargonidin, malvidin and petunidin. By way of example, metabolites canbe selected from protocatechuic acid, 2, 3, 4 trihydroxybenzaldehyde.

In one aspect, the present invention provides for a pharmaceuticalcomposition for promoting wound healing, comprising phytochemicals: a)an anthocyanin; or b) anthocyanidin or their metabolites such as C3G,PCA, 246 THBA, vanillic and hippuric acid. By way of example, theanthocyanin can be selected from cyanidin-3-glucoside ordelphinidin-3-glucoside, cyanidin-3-galactoside, andpelargonidin-3-galactoside. Also by way of example, the anthocyanidinscan be selected from cyanidin, delphinidin, pelargonidin, malvidin andpetunidin. Preferably the present composition comprises PCA and 2,4,6Trihydroxybenzaldehyde (2,4,6 THBA).

In one aspect, for example, the present disclosure provides for apharmaceutical composition comprising protocatechuic acid (PCA) wherebysaid composition reduces the growth of certain microbes, including P.acnes.

In one aspect, for example, the present disclosure provides for apharmaceutical composition comprising cyanidin-3-glucoside whereby saidcomposition reduces the growth of certain microbes, including H. pylori.

In one aspect, the present invention provides for a pharmaceuticalcomposition for treating a wound, comprising: a) an anthocyanin; b)anthocyanidin; or c) a metabolite of an anthocyanin or anthocyanidin inan effective amount whereby microbial growth is reduced. Preferably thepresent composition comprises PCA and 2,4,6 Trihydroxybenzaldehyde(2,4,6 THBA).

By way of example, the anthocyanin can be selected fromcyanidin-3-glucoside or delphinidin-3-glucoside, cyanidin-3-galactoside,and pelargonidin-3-galactoside. Also by way of example, theanthocyanidins can be selected from cyanidin, delphinidin, pelargonidin,malvidin and petunidin. By way of example, metabolites can be selectedfrom protocatechuic acid (PCA) and 2, 3, 4 trihydroxybenzaldehyde.

In another aspect, the pharmaceutical composition of this invention totreat a wound generally can include a concentration of the anthocyaninsand anthocyanidins or metabolites thereof in a concentration of at least25 mM concentration, not including the carrier. Preferably the presentcomposition comprises PCA and 2,4,6 Trihydroxybenzaldehyde (2,4,6 THBA).

In yet another example, the pharmaceutical composition of this inventionto treat a wound can include a concentration of the anthocyanins andanthocyanidins or metabolites thereof in a concentration of between 20mM to 200 mM concentration, not including the carrier. In yet otherembodiments, the pharmaceutical composition of this invention to treat awound can include a concentration of the anthocyanin, anthocyanidin, ormetabolites thereof provided in any recited composition or method of ina range of between 20 to 100 mM, not including the carrier. In anotherexample, the pharmaceutical composition of this invention to treat awound can include a concentration of the anthocyanin, anthocyanidin, ormetabolites thereof provided in any recited composition or method of ina range of between 20 to 500 mM, not including the carrier.

Further, in one example, in a pharmaceutical composition of thisinvention, PCA can be provided in a concentration approximately 50-100mM. Additionally, in one example, pterostilebene can be provided in aconcentration of approximately 35-65 mM. Further, in one example, PCAcan be provided in a concentration approximately 78 mM andpterostilebene at a concentration of approximately 40.6 mM to reducemicrobial growth or eliminate growth. Further, in one example, PCA andpsterostilbene can be provided in a combination in concentrationsprovided in this disclosure. In yet another example C3G would beprovided at a dosage of 131, 261 and 522 mg/kg.

In one aspect, the present invention provides for a pharmaceuticalcomposition for treating a wound, comprising phytochemicals: a) ananthocyanin; b) anthocyanidin; c) a metabolite of an anthocyanin oranthocyanidin such as C3G, PCA, 246 THBA, vanillic and hippuric acid.Preferably the present composition comprises PCA and 2,4,6Trihydroxybenzaldehyde (2,4,6 THBA).

The present disclosure also provides for a pharmaceutical compositioncomprising phytochemicals: a) an anthocyanin; b) anthocyanidin; c) ametabolite of an anthocyanin or anthocyanidin such as C3G, PCA, 246THBA, vanillic and hippuric acid. Preferably the present compositioncomprises PCA and 2,4,6 Trihydroxybenzaldehyde (2,4,6 THBA).

In another aspect, the present invention provides for a pharmaceuticalcomposition for promoting wound healing, comprising phytochemicals: a)an anthocyanin; b) anthocyanidin; c) a metabolite of an anthocyanin oranthocyanidin such as C3G, PCA, 246 THBA, vanillic and hippuric acid. Byway of example, metabolites can be selected from, protocatechuic acid,2, 4, 6 trihydroxybenzaldehyde. In yet another aspect, the presentdisclosure provides for a pharmaceutical composition comprisingphytochemicals: a) an anthocyanin; b) anthocyanidin; c) a metabolite ofan anthocyanin or anthocyanidin such as C3G, PCA, 246 THBA, vanillic andhippuric acid. By way of example, the metabolites are metabolitesselected from protocatechuic acid, 2, 4, 6 trihydroxybenzaldehyde.

The present disclosure provides for pharmaceutical compositions wherebythe anthocyanin, anthocyanidin, anthocyanin metabolite, anthocyanidinmetabolite, anthocyanin metabolite, or metabolites thereof, are isolatedreagents. Preferably the present composition comprises PCA and 2,4,6Trihydroxybenzaldehyde (2,4,6 THBA).

The present disclosure also provides for routes of administration of thepharmaceutical compositions, including oral, injection, intravenous,topical, sublingual, buccal, inhalation, intradermal, subcutaneous, softtissue, and cutaneous.

Oral administration of the compositions of this disclosure, includingoral gavage, may include a liquid or semisolid form, tablet, pill,capsule, powder, or gel. Preferably, oral administration will be in aliquid composition. Compositions including a liquid pharmaceuticallyinert carrier such as water may be considered for oral administration.Other pharmaceutically compatible liquids or semisolids may also beused. The use of such liquids and semisolids is well known to those ofskill in the art.

Intravenous and injection administration will be in liquid form. Otherpharmaceutically compatible liquids or semisolids may also be used. Theuse of such liquids and semisolids is well known to those of skill inthe art.

Preferably, the composition is formulated as a topical composition. Morepreferable, the vehicle of the topical composition delivery is in theform of a liquid, salve, soap, spray, foam, cream, emollient, gel,ointment, balm or transdermal patch.

In addition to the components and administration of said compositionsdisclosed above, the compositions can be in the form of an aqueoussolution. The compositions disclosed herein can also be in the form of aliquid, gel, suspension, dispersion, solid, emulsion, aerosol, forexample, powders, tablets, capsules, pills, liquids, suspensions,dispersions or emulsions. Also, the compositions disclosed herein can bein the form suitable for dilutions. Similarly, the compositions can bein the form of a powder, cream, paste, gel or solid that can bereconstituted.

Other components can be present in the composition, if desired. Forexample, the antimicrobial composition can also include at least oneadditive selected independently from a carrier, a diluent, an adjuvant,a solubilizing agent, a suspending agent, a filler, a surfactant, asecondary antimicrobial agent, a preservative, a viscosity modifier, athixotropy modifier, a wetting agent, an emulsifier, or any combinationsthereof. For example, the disclosed antimicrobial composition canfurther comprise at least one surfactant selected from a cationicsurfactant, an anionic surfactant, a non-ionic surfactant, and anamphoteric surfactant. Additionally, the disclosed antimicrobial and/orpharmaceutical compositions may further comprise medicament is selectedfrom the group consisting of burn relief medications, anesthetic agents,wound cleansers, antiseptic agents, scar reducing agents,immunostimulating agents, antiviral agents, anti keratolytic agents,anti-inflammatory agents, antifungal agents, acne treating agents,sunscreen agents, dermatological agents, antihistamine agents,antibacterial agents, bioadhesive agents, inhibitors of prostaglandinsynthesis, antioxidants, and mixtures thereof.

Also, the disclosed antimicrobial compositions can optionally includeone or more additives such as carriers, adjuvants, solubilizing agents,suspending agents, diluents, surfactants, other antimicrobial agents,preservatives, fillers, wetting agents, antifoaming agents, emulsifiers,and additives designed to affect the viscosity or ability of thecomposition to adhere to and/or penetrate the wound.

In one embodiment, the disclosed antimicrobial compositions, includingthe selected active components, including the anthocyanins oranthocyanidins and metabolites thereof, are without causing significantundesirable biological effects or interacting in a deleterious mannerwith any of the other components of the composition in which it iscontained.

In one embodiment, the disclosed compositions, including the selectedactive components, including the anthocyanins or anthocyanidins andmetabolites thereof, are provided as a nutraceutical and provided as adietary supplement without causing significant undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the foodstuff in which it is contained.

In one aspect, the antimicrobial composition provided herein, includinganthocyanins or anthocyanidins and metabolites thereof, are used inagricultural settings, including but not limited to nurseries,commercial farming, agricultural research facilities, residentialgardens and produce processing facilities, and are applied to plants andtrees to inhibit, reduce or substantially eliminate microbial bioburdenas well as many fungal bioburden on plants, trees, and surfaces thereof,including leaf surfaces.

In another embodiment, antiseptic compositions of the present inventionare formulated for use in liquids, solutions, gels, soaps, creams,powders salves and other preparations designed for topical use asantiseptic agents, sprays, foams, antibacterial treatments, wipes andthe like. In another embodiment, antiseptic compositions of the presentinvention are formulated as a hand antiseptic.

In yet another embodiment, antiseptic compositions of the presentinvention are used in industrial settings such as in water treatmentfacilities, including swimming pools or water treatment plants, foodpreparation, including but not limited to poultry and fish processingfacilities or produce handling and packaging settings to inhibit, reduceor substantially eliminate microbial bioburden, as well as many fungalbioburden. In addition to adding the antiseptic composition to a watersupply or water supply system, industrial equipment and surfaces may becontacted with, or soaked in, the antiseptic compositions of the presentinvention.

In yet another embodiment, sanitizing compositions of the presentinvention are formulated for use in liquids, solutions, gels, soaps, andother preparations designed for use as sanitizing agents, liquids,including sprays, foams, gels, soaps, sanitizing treatments, and thelike when used as a sanitizing solution, including but not limited to,use in food processing facilities, including food-processing equipmentand utensils, and on other food-contact articles.

In yet another embodiment, sanitizing compositions of the presentinvention use in food processing facilities, including food-processingequipment and utensils, and on other food-contact articles areformulated to include any components generally recognized as safe foruse in food processing facilities, including but not limited to, aqueoussolutions containing potassium, sodium or calcium hypochlorite, asolution of hydrogen peroxide, an aqueous solution containing potassiumiodide, sodium lauryl sulfate, sodium-toluenesulfonchloroamide,solutions containing dodecylbenzensulfonic acid, other acceptabledetergents and the like.

In one aspect, the one or more of the additives can be an agent that isacceptable when used in or on foods and beverages and which can beconsumed by a mammal (e.g., human, pet, livestock, etc.) along with theselected active components, including the anthocyanins or anthocyanidinsand metabolites thereof, without causing significant undesirablebiological effects or interacting in a deleterious manner with any ofthe other components of the composition in which it is contained.

In another aspect, the compositions of the present invention, includingthose compositions comprising: a) an anthocyanin; or b) ananthocyanidin; or c) a metabolite of an anthocyanin metabolite; or d) ametabolite of an anthocyanidin metabolite, or e) a combination thereof,are used in food processing, packing, manufacturing, handling,preparing, treating, transporting or holding as a food additive withoutcausing undesirable effects or interacting in a deleterious manner. Byway of example, protocatechuic acid can be used as an additive in meat,including the handling and processing, without causing undesirableeffects or interacting in a deleterious manner with the meat.

In yet another aspect, the compositions of the present invention,including those compositions comprising: a) an anthocyanin; or b) ananthocyanidin; or c) a metabolite of an anthocyanin metabolite; or d) ametabolite of an anthocyanidin metabolite, or e) a combination thereof,are used in food processing, including cold sterilization of foodcontainers, including bottles, without causing undesirable effects orinteracting in a deleterious manner.

In other examples, the antimicrobial compositions disclosed herein canfurther comprise a carrier. The term “carrier” means a compound,composition, substance, or structure that, when in combination with acompound or composition disclosed herein, facilitates preparation,administration, delivery, effectiveness, or any other feature of thecompound or composition. Examples of carriers include water, isopropylalcohol ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol,and the like), vegetable oils, and suitable mixtures thereof.“Pharmaceutically acceptable carrier” means a compound, composition,substance, or structure that is useful in neither preparing apharmaceutical composition which is generally safe, non-toxic, andneither biologically nor otherwise undesirable and includes that whichis acceptable for veterinary as well as human pharmaceutical use.

In a further example, the antimicrobial compositions disclosed hereincan also comprise adjuvants such as preserving, wetting, emulsifying,suspending agents, and dispensing agents. Prevention of the action ofother microorganisms can be accomplished by various antifungal agents,for example, parabens, chlorobutanol, phenol, and the like.

Suitable suspending agents can include, for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, or mixtures of these substances, and the like.

The disclosed antimicrobial compositions can also comprise solubilizingagents and emulsifiers, as for example, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl alcohol,benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide,oils, in particular, cottonseed oil, groundnut oil, corn germ oil, oliveoil, castor oil and sesame oil, glycerol, tetrahydrofur fury 1 alcohol,polyethyleneglycols and fatty acid esters of sorbitan or mixtures ofthese substances, and the like. The additives can be present in thedisclosed compositions in any amount for the individual anthocyanin oranthocyanidin compound components.

EXAMPLES Example 1: Use of In Vitro Studies for AntimicrobialSusceptibility Testing of Anthocyanins, Anthocyanidins, or Metabolitesand Compounds Thereof

This example describes the method for testing the antimicrobialsusceptibility of anthocyanins, anthocyanidins, or metabolites andcompounds thereof. The Kirby-Bauer method of disc diffusion was used fortesting, following a standard set of procedures recommended by theNCCLS. In this methodology, a set of discs saturated with either testingcompounds or a control was placed on inoculated agar plates. The plateswere inoculated with organisms listed in the tables provided in FIG. 6,including C difficile, P. acnes, C. prefringens, L. casei, C. albicans,E. coli, ATTC 8739 and ATCC 43895, S. aureus, S. mutans, S. pyogenes, P.aeruginosa and K. pneumonia. The control sample was amoxicillin, anantimicrobial with very effective broad spectrum antibiotic properties.Samples included delphinidin, pelargonidin, cyanidin CI, 28%cyanindin-3-glucoside (C3G), protocatechuic acid (PCA) and 2,4,6Trihydroxybenzaldehyde (2,4,6 THBA).

After 18, 24, or 48 hours of incubation, depending upon themicroorganism, each plate was examined. The diameters of the zones ofcomplete inhibition were measured, including the diameter of the disc.Zones were measured to the nearest millimeter, using sliding calipers.The size of the zones of inhibition was interpreted by referring toNCCLS standard. Results were interpreted as follows: NI was noinhibition of growth under the test sample, I was inhibition of growthunder the test sample, NZ indicated no zone of inhibition surroundingthe test sample, and CZ indicated a clear zone of inhibition surroundingthe sample and zone width in millimeters. See FIG. 6 for completeresults.

Results

Referring to FIG. 6 and FIG. 32, the testing samples had bactericidaland bacteriostatic activity against many of the organisms. Of note, P.acnes, an organism that is very difficult to treat, often requiringmultiple current antibiotics for effective treatment, was susceptible toboth C3G and PCA. Indeed, both of these test samples were bactericidalagainst P. acnes. Additionally, PCA was also effective againstStaphlococcus aureus ATCC 33591, known as Methacillin Resistant StaphAureus (MRSA), Staphlococcus epidermidis ATCC 51625, known asMethacillin Resistant Staph Epidermidis (MRSE), E. coli 8739 and 43895,and Legionella 43662.

PCA was also shown to have some effectiveness against Pseudomonasaeruginosa, a common pathogen in wounds, especially burns, as well aschronic lung infections. Amoxicillin, the control sample, had no effecton P. aeruginosa. Similarly, Candida albicans, frequently a co pathogenin wounds, was susceptible to PCA.

PCA was also shown to have some effectiveness against Pseudomonasaeruginosa, a common pathogen in wounds, especially burns. Amoxicillin,the control sample, had no effect on P. aeruginosa. Similarly, Candidaalbicans, frequently a co pathogen in wounds, was susceptible to PCA.

In summary, the present invention provides advantages over the priorart, including providing anthocyanin, anthocyanidin, their metabolitesor combinations thereof to a wound to provide a reduction or eliminationof bacteria. It is contemplated that the invention will also find use inthe treatment of surfaces, including medical devices and medicalimplants, to reduce or eliminate bacteria.

Example 2: Use of Mouse Model to Determine Dose Levels and Intervals ofTest Samples

Methods:

Mice had back skin tape stripped and the stripped site (wound) wasinfected with P. aeruginosa (ACTA 9027). The test reagents were appliedtopically in an aqueous solution on the stripped site at two hours anddaily for four days.

Cyanidin 3-glucoside (C3G), an anthocyanin, and its main metabolite PCAwere formulated and tested at several doses. The aqueous carrier waswater. The C3G formulation included 50 mM, 100 mM and 200 mM doseconcentrations. Similarly, the PCA formulation included at 50, 100 and200 mM dose concentrations.

Results

Results were collected from the mice at day five. Both C3G and PCAdecreased the bacterial burden; however, none were statisticallysignificant. See FIG. 9A. There was a trend towards a decreasingconcentration of PCA, with 50 mM being the most effective. The mosteffective dose of C3G was 100 mM. It is contemplated that because C3Gdegrades to PCA in this environment, the test results may indicate thatC3G was not being tested alone, but rather was a combination of C3G andits metabolites, including a combination of C3G and PCA as the effectiveagents.

Example 3: Use of Mouse Model to Further Determine Effective Dose Levelsand Dose Intervals of Test Samples

Methods:

Mice had back skin tape stripped and the stripped site (wound) wasinfected with P. aeruginosa (ACTA 27853). The test reagents were appliedtopically in an aqueous solution on the stripped site at two hours anddaily on day 1, 2 and 3.

C3G, an anthocyanin and its main metabolite PCA were formulated andtested at several doses. The aqueous carrier was water. The C3Gformulation included 100 mM and 200 mM dose concentrations and the PCAformulation included 25 and 50 mM dose concentrations.

Results

Results were collected from the mice at day two and four. Both C3G andPCA decreased the bacterial burden at 48 and 96 hours. (See FIG. 9B).The most significant decrease of bacteria was observed at 25 mM of and100 and 200 mM of C3G. Although PCA at 25 mM reduced the bacterialburden at both time periods, its activity was statistically significantat 48 hours. C3G at both 100 mM and 200 mM significantly reduced thebacterial burden at 48 and 96 hours.

Example 4: Use of a Mouse Model for Wound Healing

Methods:

Mice were shaved but unstrapped and uninfected (normal rodent skin). Thetest reagents were applied topically in an aqueous solution on theunstripped site at two hours and daily on day 1, 2 and 3.

Testing reagents consisted of C3G and PCA formulated at one dose, 100 μMin an aqueous solution.

Results:

Referring to FIG. 10, there was little or no stimulation of IGF-1 andTGF-β at local levels observed at the 100 μM concentration of testingreagents. In fact, levels of EGF actually decreased below normal levels.There was observed a decrease of all three local growth hormones at 100uM of C3G. These results suggest that mice skin differs in response to adose that has been shown to stimulate human synovium to produce IGF-1.Thus, this low of a dose is not useful for rodents for this purpose.

Example 5: Use of Mouse Model to Determine Isolated Effect of 25 mMSolution of PCA in Various Environments

Methods:

Four different conditions were used: mice had back skin tape strippedand the stripped site (wound) was infected with P. aeruginosa; mice hadback skin stripped and were not infected, mice had taped stripped,infected and treated with PCA, mice were tape stripped, uninfected, andtreated with PCA. When used, the PCA test reagent was applied topicallyin an aqueous solution on the stripped site at two hours and 24 hours.

The testing reagents consisted of and PCA formulated at one dose, 25 mM,in an aqueous solution. Levels of IGF-1, TGF-β, and EGF levels in theskin tissue at 48 hours were measured by ELISA. There were two controlgroups; the stripped skin and the stripped skin and infected.

Results:

Referring to FIG. 13, the infected stripped skin showed the highestlevel with IGF-1 (statistically significant) and TGF-β. This isrepresentative of tissue response to injury and infection; similarly,the EGF response was very inconsistent compared to the other two growthhormones.

The EGF response levels were different than either IGF-1 or TGF-β. Theywere highest in the stripped and uninfected wound and lowest in thestripped, infected and treated wound. Therefore, the treatment optimizedthe amount of hormone production compared to the untreated infection.This is beneficial to limit scarring while promoting healing over thecontrols. Overall, PCA at 25 mM acts on stripped and infected mice skinand optimizes the IGF-1 production and optimizes the local growthhormones.

Example 6: Use of Mice to Establish Wound Promoting Effect ofCompositions

Method:

Fifteen rodents were used to establish the histological findings ofstripped skin, stripped and infected skin, and stripped, infected andtreated wound. There were two control groups and four experimentalgroups according to the following:

Control Group 1: three mice with only tape stripped wounds on the back.These mice were not infected or treated. The skin was harvested at timezero, 2 and 48 hours for histology examination.

Control Group 2: three had tape stripped wounds and infection. Tissuesubmitted at 2 and 48 hours for histological examination.

Experimental Groups: There were 4 experimental groups. In these groups,mice had skin stripped wounds and infection. Treatment varied by reagentand dosage. Testing reagents included PCA at 25 at 25 and 50 mM and C3Gat 100 and 200 mM.

Pseudomonas aeruginosa (ATCC 27853) procured from American Type Culture

Collection, Manassas, Va. was used to infect the experimental groups ofmice. The organism was grown overnight at 37° C. at ambient atmospheretrypticase soy agar plates supplemented with 5% sheep blood cells. Theculture will be aseptically swabbed and transferred to tubes oftrypticase soy broth. The optical density will be determined at 600 nm.The cultures will be diluted to provide an inoculum of approximately 9.0log₁₀ CFU per mouse in a volume of 100 μL. Inoculum count was estimatedbefore inoculation by optical density and confirmed after inoculation bydilution and back count.

The testing reagents were topically applied at 2 and 24 hours with 100uL of fluid spread over the wound.

The following histological assessments were conducted:

Surface Cellularity: The histological assessment included the presenceor absence of the surface cellularity and the depth of the cells.

Dermis: vascularity and inflammation.

Thickness: The thickness of the dermal layer was observed.

Hair Follicles: The hair follicles and the layer of surrounding cellswere observed. Hair follicles presence is critically important to skinwound healing. (Gharzi A, Reynolds A J, Jahoda C A. Plasticity of hairfollicle dermal cells in wound healing and induction. Exp Dermatol. 2003April; 12 (2):126-36). The dermal sheath surrounding the hair folliclehas the progenitor cells for contributing fibroblasts for wound healing.(Johada C A, Reynolds A J. Hair follicle dermal sheath cells: unsungparticipants in wound healing. Lancet. 2001 Oct. 27; 358(9291):1445-8).

Vascularity: Vascularity was observed, but an assessment of angiogenesiswas not performed on the 48 hour material since new vascularity takesthree to twelve days to develop. (Busuioc C J, et al. Phases ofcutaneous angiogenesis process in experimental third-degree skin burns:histological and immunohistochemical study. Rom J Morphol Embryol. 2013;54(1):163-710.)

Inflammation: The presence of cellular infiltration was observed and itslocation.

Skin Thickness: The thickness of the skin was estimated related to theuninfected, untreated wound. This depth was estimated on the uniformhistology photomicrographs from the surface to the muscle layer.

Results:

The following results were observed in each group:

CONTROL GROUP 1: Uninfected and Untreated.

Time Zero: (See FIGS. 14-15) At time zero following the wound strippingthere was cellular covering of the surface. The dermal layer was notthickened. The hair follicles have a single cellular lining. There wasminimal vascularity and no inflammation. The depth of the tissue wasconsidered zero for future bench mark. 0+

2 hours: (See FIGS. 16-17) At 2 hours following the wound stripping thesurface remained covered with cellularity. The dermal layer wasminimally thickened. The follicles and cellular lining was the same.There was minimal increase in vascularity and inflammation. The increasein the depth of the tissue was considered 0.5+.

48 hours: (See FIGS. 18-19) At 48 hours the wound stripped, uninfected,untreated specimens showed natural history response of surface cellularproliferation and thickness. The dermal layer was thickened. The hairfollicles were present with single layer cellular lining. Thevascularity was increased in amount compared to the 2 hour specimens.The inflammation was present throughout the dermis and muscle layer. Thethickness was considered 0.5+.

CONTROL GROUP 2: Infected and Untreated.

2 hours: (See FIGS. 20-21) The histological assessment showed the woundstripped, infected, but untreated controls at 2 hours to have multiplecellular covering on surface. There was minimal thickening of the dermallayer. The hair follicles were abundant and had double layer cellularlining. There was minimal vascularity and no inflammation in thespecimens. The thickness was assigned 0.5+.

48 hours: (See FIGS. 22-23) At 48 hours the surface cellular coveringwas gone. The dermal layer had minimal thickening. The hair follicleswere present, with minimal cellularity lining. There was marked increasein vascularity and minimal inflammation in dermis layer. The depth wasconsidered 0.5+ compared to time zero.

EXPERIMENTAL GROUP PCA 25 mM

48 hours: (See FIGS. 24-25) The cellular covering of the surface wasabundant and multiple cell layers. The dermal layer was thickened. Thehair follicles were prominent with multiple cellular lining. There wascollagen proliferation between the epidermis and dermis. Additionally,there was moderate vascularity, but less than that seen in infecteduntreated group. There was abundant inflammation and it was greater thanwas seen in the PCA 50 dose. Thickness was assigned 2+.

EXPERIMENTAL GROUP PCA 50 mM

48 hours: (See FIGS. 26-27) The surface was covered with multiple layersof cells. The dermal layer was thicker. The hair follicles had doublelayer of cells. There was increased vascularity. Inflammation alsoincreased in the dermis and below the muscle layer. The tissue thicknesswas assigned 2+.

EXPERIMENTAL GROUP C3G 100 mM

48 Hours: (See FIGS. 28-29) There was multiple cellular covering of thesurface. The dye of the C3G was apparent on the skin surface indicatingit had not changed color due to pH nor completely degraded. The dermallayer was thicker. The hair follicle had single and double cellularlining. The vascularity was prominent. There was inflammation in thedermis and muscular layer and below. The thickness of the tissue wasassigned 2+.

EXPERIMENTAL GROUP C3G 200 mM

48 Hours: (See FIGS. 30-31) There was evidence of the C3G materialremaining on the skin surface. The surface cellular layer was multiplecells thick. The dermal layer was thickened. The hair follicles hadsingle and double cellular lining. The vascularity was increased. Therewas inflammation in the dermis and muscular layer. The thickness wasassigned 2+.

These results confirm that an anthocyanin (˜38% C-3-G as the source) andthe main metabolite of anthocyanins and anthocyanidins, protocatechuicacid (PCA) when applied topically at various calculated doses to thestripped skin wound of a rodent were bactericidal in 48 to 96 hours.There was a 10,000 fold kill of Pseudomonas aeruginosa in 48 hours withboth reagents and dose.

The results also show by histology a simultaneous healing of theexperimentally created wound in the same time frame. C-3-G and PCA intwo different doses stimulated tissue repair as evidence by histology.

Specifically, the experimental model provided evidence of a histologicalcontrast between the control and experimental groups. At 48 hours,Control Group 2 that was wound stripped and infected showed a clearcontrast to the uninfected Control Group 1. In the skin strippedinfected group there was loss of the epithelial cellular covering, nofollicular cellular proliferation, marked increase in vascularity andlittle inflammatory response. This histological condition provided clearcontrast to the treatment groups. All treatment groups by comparisonshowed healing response with multiple layer cellular proliferation onthe surface, multiple layer cellular proliferation along the hairfollicles, less vascularity, but an inflammatory cellular response inthe dermis and muscular levels. See FIGS. 14-31. PCA at a concentrationof 25 mM also showed collagen layer formation between the epidermis anddermis. (See figures/photos 24 and 25). This response is beneficial inthe use of anthocyanin and anthocyanidins and metabolites thereof as acosmetic agent to promote wound healing and improve skin health,including wrinkle reduction or removal. This method of use ofanthocyanin and anthocyanidin metabolites, and particularly PCA, isbased upon the two fold response; the collagen layer increase and theskin swelling that increased the depth of the skin.

Example 7: PCA's Effect on Pseudomonas Aeruginosa ATCC 700888 andStaphylococcus aureus ATCC 33591 (MRSA) Biofilms

The inventor has shown that a composition comprising PCA was able tostop the growth of a biofilm formation as well as destroy already formedbiofilms. The biofilms tested were Pseudomonas Aeruginosa ATCC 700888and Staphylococcus aureus ATCC 33591 (MRSA).

The following amounts were tested on a polyester cloth and sintered 316stainless steel mesh: The cloth was a piece cut from a polyester pillowcase. The cloth was soaked in the PCA solution and air dried for 24hours. The cloth was dried when tested.

-   1: 20 grams of PCA in 100 ml of 70% isopropyl alcohol, vehicle:    cloth material, 1-1-   2: 20 grams of PCA in 100 ml of 70% isopropyl alcohol, vehicle:    cloth material, 1-2-   3: 20 grams of PCA in 100 ml of 70% isopropyl alcohol, vehicle:    3-ply sintered mesh, 1-1-   4: 20 grams of PCA in 100 ml of 70% isopropyl alcohol, vehicle:    3-ply sintered mesh, 1-2-   5: 20 grams of PCA in 100 ml of 70% isopropyl alcohol, vehicle:    5-ply sintered mesh, 1-1-   6: 20 grams of PCA in 100 ml of 70% isopropyl alcohol, vehicle:    5-ply sintered mesh, 1-2-   7: Glass Slide (to serve as control article), 1-1-   8: Glass Slide (to serve as control article), 1-2-   9: PCA crystals imbedded, vehicle: 3-ply sintered 40 micron mesh,    1-1-   10: PCA crystals imbedded, vehicle: 3-ply sintered 40 micron mesh,    1-2-   11: PCA crystals imbedded, vehicle: 5-ply sintered 40 micron mesh,    1-1-   12: PCA crystals imbedded, vehicle: 5-ply sintered 40 micron mesh,    1-2

The bacteria (Pseudomonas Aeruginosa ATCC 700888 and Staphylococcusaureus ATCC 33591 (MRSA)) were placed in reactors and allowed to growand form biofilms. Then cloths and metal were treated by coating withPCA solutions and then were left to dry. Two sets of the stainless steelmesh had crystals imbedded into the mesh to replicate placement into amesh or coated joint implant. A standard ASTM E-2647 drip flow biofilmreactor was used to grow a biofilm and the treated surfaces (as well asthe control) were placed into the reactors and the biofilm was allowedto grow for about 6 hours. The samples received a continuous nutrientflow for an additional time period for about 48 hours to promote asteady growth rate of the biofilm. Then the biofilm was removed,analyzed and a microbial count and log density measurements were takenfor each sample. Colony forming unites (“CFU”) were counted (which is anestimate of the number of viable bacterial. Log density is thecalculation of the biofilm present.

It was found that A 10% concentration as not as effective againstPseudomonas, but a 20% concentration of PCA was very effective. SeeFIGS. 33 and 34. FIG. 34 shows that the materials that were treated hada very much smaller log density and CFUs than the control material(glass slide).

It was found that a 30% concentration of PCA against MRSA was veryeffective. See FIGS. 35 and 36.

Example 8: Spray on Solution of PCA and Time Study of PCA's Effect onPseudomonas Aeruginosa ATCC 700888 and Staphylococcus aureus ATCC 33591(MRSA) Biofilms

Next the time necessary to destroy biofilms and kill bacteria wastested. A biofilm consisting of over 10 million organisms were formed ona glass slide. A single spray of 30% PCA mixed with isopropyl alcoholwas applied to the glass slide. The colony forming units (CFU) wereexamined at 30 minutes and 60 minutes after the single spray.Incidentally, typical tests for testing the ability of an antibiotic towork are done for 48 hours. The tests performed were as follows:

Glass slides were inoculated at Time 0. Batch phase was performed for 6hours to allow for biofilm formation on the glass slides. The drip flowmechanism was then turned on to provide a continuous flow of nutrientsto the glass slides over 48 Hours. After 48 hours, 2 sets of glassslides were sprayed with a 30% PCA solution. One set was removed andanalyzed for biofilm reduction after 30 minutes. The other set wasremoved after 60 minutes.

2 sets of control slides were also removed and analyzed after 30 minutesand 60 minutes. The control slides were not treated with 30% PCA andwere used for comparative purposes.

The Log Reduction Calculations were performed as follows: The mean 30%PCA treated samples were compared to the mean positive control samples,per time point evaluated.

For Pseudomonas aeruginosa, after 30 minutes of the application of the30% PCA spray, there was a 3.3 log reduction. After 60 minutes, therewas 2.2 log reduction, which amounts to a 99.9% reduction in the numbercolony forming units. See FIG. 37.

For Staphylococcus aureus, after 30 minutes of the application of the30% PCA spray, there was a reduction of 60 million CFUs to 3 million andafter 60 minutes, there was reduction, of 25 million to 2 million CFUs.See FIG. 38.

Thus, the results show that a 30% PCA spray killed 90% of the biofilmsin 30 minutes. The FDA only requires a 90% reduction.

It is noted that this above experiment was chosen to replicate aclinical condition, involving a metal or linen implant even to theextreme because there would never be that concentration of biofilms norbacteria in practice flowing over the implants. Accordingly, theinvention also provides a method of blocking initial attachment of thebacteria to the implant and therefor preventing growth/development of abiofilm on an implant.

Example 9: Testing Against Propionibacterium acnes

BALB/c mice were infected with Propionibacterium acnes via intradermalinjection and treated topically with varying concentrations of a noveltest compound, PCA, at 2, 24, 48 and 72 hours following challenge.Efficacy was evaluated by CFU analysis from skin samples harvested at 96hours post challenge.

These data demonstrate that P. acnes establishes a steady intradermalcolonization in the skin of BALB/c mice. When administered topically,PCA at 60 mg/kg, demonstrated a bacteriostatic effect and reduced P.acnes CFU burden in mouse skin by a statistically significant amount.All lower amounts of PCA showed no such effect.

Female BALB/c mice, ordered from Harlan and weighing 17-19 g, wereacclimated to housing conditions and handled in accordance with AUPnumber TP-18-13. The animals were acclimated for 4 days prior tobacterial challenge. Only animals deemed healthy and fullyimmunocompetent were included in this study. Cages were prepared with 2mice per cage.

The animals were fed Teklad Global Rodent Diet (Harlan) and water adlibitum. Mice were housed in static cages with Teklad ⅛″ corn cobbedding inside bioBubble® Clean Rooms that provide H.E.P.A filtered airinto the bubble environment at 100 complete air changes per hour. Alltreatments and infectious challenges were carried out in the bioBubble®environment. The environment was controlled to a temperature range of74°±4° F. and a humidity range of 30-70%. Treatment groups wereidentified by cage card. All procedures carried out in this experimentwere conducted in compliance with all the laws, regulations andguidelines of the National Institutes of Health and with the approval ofthe TransPharm Animal Care and Use Committee.

Bacterial Cultures

Propionibacterium acnes (1100; ATCC 6919), procured directly from theAmerican Type Culture Collection.

Skin Preparation

On Day −1, each mouse was anesthetized in an Isoflurane inductionchamber and the lesion site was cleared of hair. An area ofapproximately 2.0 cm×2.0 cm of skin on the dorsal area of each mouse wascleared through use of the depilatory agent Nair®.

Challenge

Cultures were grown for 96 hrs at 37° C. in an anaerobic atmosphere onTS agar plates supplemented with 5% sheep blood cells. The culture wasaseptically swabbed and transferred to tubes of TS broth and allowed togrow for 72 hours. The cultures were diluted to provide challengeinoculum of approximately 6.0-7.0 log 10 CFU per 50 μL in PBS. On Day 0each mouse was anesthetized using Isoflurane. Each animal on the studywas administered 50 μL of the bacterial suspension via intradermalinjection in the dorsal area that was previously denuded of hair. Thefinal CFU count from the challenge suspension determined that 6.0 log 10CFU per mouse were delivered.

Formulation and Dosing

The test treatment, PCA, was provided by the study Sponsor andformulated using sterile water. Treatments were administered topicallyin a dose volume of 0.1 mL. Treatments were given at 2, 24, 48 and 72hours post challenge at 60 mg/kg (78 mM; Group 3), 30 mg/kg (39 mM;Group 4) or 15 mg/kg (19.5 mM; Group 5).

TABLE 1 Animal Challenge, Treatment and Harvest Schedule Intradermal CFUGroup n P. acnes Treatment ROA Schedule harvest* 1 2 6.0 log untreatedNA NA  2 hr 2 2 6.0 log untreated NA NA 96 hr 3 2 6.0 log PCA 60 mg/kgTopical 2, 24, 48, 72 96 hr hrs 4 2 6.0 log PCA 30 mg/kg Topical 2, 24,48, 72 96 hr hrs 5 2 6.0 log PCA 15 mg/kg Topical 2, 24, 48, 72 96 hrhrs *Relative to Challenge at 0 hrResults and DiscussionInfection/Treatment/General Observations

None of the study subjects displayed any acute adverse events associatedwith the treatments. None of the test subjects succumbed to theinfection or showed signs of morbidity, which could be attributed topenetration of the infection into the circulatory system or deep tissue.No treatment group displayed adverse signs beyond those expected formice which have received a superficial bacterial infection.

The test article preparations were administered topically at 2, 24, 48and 72 hours following the bacterial challenge. While untreated micewere harvested at 2 hours post infection, CFU burden was not detected.However at 96 hours post infection, the CFU burden rose from 6.0 log 10to 6.65 log 10, indicating a successful inoculation.

At 96 hours following challenge, mice were humanely euthanized and skinwas aseptically removed from the infection site. Skin samples wereplaced in homogenation vials with 2.0 mL PBS, weighed and homogenizedusing a mini-bead beater. Homogenate was serially diluted and platedanaerobically on TSA agar plates for enumeration of colony forming unitsper gram of skin tissue.

The mean bacterial burden of the untreated group at 96 hours was 6.65log 10. CFU levels in all treated groups were compared to the untreatedgroup to determine statistical significance. Only the high dose of PCA(60 mg/kg) showed significant reduction of CFU burden when compared tothe untreated control (P=0.0285). That an approximately 1 log 10reduction was observed indicates that at this concentration PCA, isbacteriostatic and not bactericidal. All other treatments werestatistically non-different than the untreated control (FIG. 39).

These data demonstrate that P. acnes establishes a steady intradermalcolonization in the skin of BALB/c mice. When administered topically,PCA at 60 mg/kg (78 mM solution), demonstrated a bacteriostatic effectand reduced P. acnes CFU burden in mouse skin by a statisticallysignificant amount. All lower amounts of PCA showed no such effect.

Example 10: Antibiotic Testing with PCA or 246 THBA Using PropyleneGlycol

PCA or 246 THBA were combined with propylene glycol (PPG). The PPG wasplaced on a paper disc and then either PCA or 246 THBA was applied. Thepaper disc was then placed on colonies of various bacterial in a Petridish. At a certain uniform time they were inspected and classified inthe following categories:

-   -   NI: no inhibition of bacteria growth under the sample    -   I: inhibition of bacterial growth under the sample    -   NZ: no accompanying zone of inhibition    -   CZ: clear zone of inhibition surrounding the sample and zone        measured in millimeters (mm).

The results were as follows:

-   Water/PCA/Klebssilla pneumonia ATCC4352: I/CZ/2 mm-   PPG/PCA/Klebssilla pneumonia ATCC4352: I/CZ/3 mm-   Water/PCA/Pseudomonas aeruginosa ATCC9027: I/CZ/10 mm-   PPG/PCA/Pseudomonas aeruginosa ATCC9027: I/CZ/4 mm-   Water/246 THBA/Pseudomonas aeruginosa ATCC9027: I/CZ/2 and 3 mm-   PPG/246 THBA/Pseudomonas aeruginosa ATCC9027: I/CZ/3 mm-   Water/246 THBA/Staphylococcus aureus ATCC33591 (MRSA): I/CZ/14 and    15 mm-   PPG/246 THBA/Staphylococcus aureus ATCC33591 (MRSA): I/CZ/12 mm

Example 11: PCA to Sterilize/Disinfect Human Skin Study

A randomized double blinded study was performed at Loma Linda MedicalSchool. It involved 4 phases over two years' time. The methods were asfollows:

Phase 1: The active test reagent was topically applied 1.54% PCA insterile water to the anterior shoulder region. This 1.54% solution ofPCA in water was used effectively in our prior animal wound studies. Thecontrols were Chloroprep (2% Chlorhexidine in 70% isopropyl alcohol) andBetadine (9.0% to 12.0% available iodine in water). Cultures were takenbefore application and 20 minutes after application. The initial harvestwas by a surface swab. Application was by soaked sponge, without forceor scrubbing. The second harvest was performed with the back edge of asterile knife blade scraping with pressure in attempt to maximize theharvest from the deeper sebaceous glands and hair follicles. Thespecimens were placed in culture media. Bacteriology was performed atWuXiAppTec in Marrietta, Ga.

Phase 2 included eleven medical students and was same method as Phase 1.However the PCA vehicle was changed to 70% isopropyl alcohol. Thisallowed a higher concentration of PCA than possible in sterile water,10%. Phase 2a: The 70% isopropyl alcohol vehicle was tested for itsbactericidal properties. All cultures that were negative or markedlyreduced with PCA topical solution were examined for exact nature of theindex bacteria and the post treatment cultures that showed no or minimalgrowth. In this way it was learned what specific bacterial strains PCAcould eliminate or reduce.

Results:

Phase 1 showed the aqueous solution of 1.54% PCA to be partiallyeffective as compared to the controls. In phase 1 of the Loma LindaStudies, 1% PCA in water showed no growth in 7 of 22 subjects on aerobicculture and 10 cultures showed reduced growth. By anaerobic culture, 6of 22 cultures showed no growth and 15 showed reduced colony growth. Sixheavy growth pre-treatment cultures prior to the 1.24% PCA treatmentwere chosen to examine the isolates to learn what pathogens were killedor not killed. Most of the bacteria eradicated were non pathogens.Sample culture #29 showed 5 unique colonies by aerobic culture and 2unique colonies by anaerobic culture. After 1.24% PCA treatment therewas no growth on either culture. Therefore pre-treatment cultures wereexamined for the species. The chart below is the result of the specificspecies colony identified and the method of identification used. Theorganisms were predominately non pathogens except for P. acnes. Howeverall of which were removed by the treatment.

GRAM STAIN & ORGANISM METHOD OF SAMPLE ID CELL MORPHOLOGY IDENTIFICATIONIDENTIFICATION 1(A) Gram positive cocci Micrococcus luteus/lylaeBiochemical Analysis 1(B) Gram positive cocci Micrococcus luteus/lylaeBiochemical Analysis 1(C) Gram positive cocci Micrococcus luteusBiochemical Analysis 1(D) Gram positive cocci Micrococcus luteusBiochemical Analysis 1(E) Gram positive cocci Staphylococcus epidermidisDNA Sequencing 2(A) Gram positive cocci Staphylococcus capitisBiochemical Analysis 2(B) Gram positive rods Propionibacterium acnes DNASequencing

Two other index pre-treatment cultures showed heavy growth ofPropionibacterium acnes, a potential pathogen. The post treatmentcultures showed the colonies of P. acnes was decreased to 5 colonies inone and 1 colony in another, but not eliminated. This suggested that PCAmay be effective against P. acnes if a higher concentration was appliedin subsequent Phases of this study. Testing with 1.24% in water killedbacteria of normal flora as shown above chart. To increase efficacy andto improve skin penetration to the depth of the hair follicles that canharbor bacteria, Phase II of the study used 70% isopropyl alcohol (30%water) 85 ml so as to increase the concentration of PCA.

Phase II: This study involved 11 human subjects. There were 5 Males and6 females. The ages were 23-33 years. There were two reagents. Thecontrol was 70% isopropyl alcohol. The PCA source was a phytochemicalextract from Nanjing Zelang Medical Technology Co. LTD. This source waschosen due to markedly reduced cost of goods compared to that which isbiochemically manufactured. The experimental dose was 9+/−% PCA in 70%isopropyl alcohol. 10 grams of PCA was placed in 100 ml of isopropylalcohol. The isopropyl alcohol allowed for a greater dose of PCA thanwater. (allowed more PCA to be dissolved). The initial harvest was by asurface swab. Application was by soaked sponge, without force orscrubbing. The second harvest was performed with the back edge of asterile knife blade scraping with pressure in attempt to maximize theharvest from the deeper sebaceous glands and hair follicles.

Cultures were stored in ice and shipped in FedEx ice box to WuXiapptecin Marietta, Ga. for aerobic and anaerobic cultures and held for 21days. Method used for bacterial species identification was Vitek®MS-MALDI-TOF MS. It was determined that the dose or concentration of PCAneeded to be increased, which required changing the water to 70%isopropyl alcohol. This is the same as used with Chloroprep® which is 2%w/v chlorhexidine gluconate in 70% isopropyl alcohol. 10% PCA in 70%isopropyl alcohol was compared to the control 70% isopropyl alcohol inPhase II. The results of Phase II showed the following summary of the nogrowth culture results after treatment.

Aerobic Anaerobic PCA/IP alcohol 10/11 no growth  9/11 no growth ControlIP alcohol 6/11 no growth* 4/11 no growth *Note that 3/11 of the 70% IPAgroups showed increased colony growth after IPA treatment alone.

Phase 2 results with 9+% solution of PCA were compared to similar testPhase 1 and this showed this to comparable to Betadine in effectiveness,but not with Chloroprep, which killed all the bacterial colonies. ThisPCA/70% isopropyl alcohol solution was effective against 10/11 indexcultured aerobic bacteria and reducing on anaerobic culture in 9 of 11subjects. The two showing residual colonies were one (n=1) colony ofPropionibacterium acnes in each of the two cultures. Phase 2a showedthat 70% isopropyl alcohol (IPA) alone had few antibacterial properties.Of course sterile water used in Phase 1 had no antibacterial properties,therefore the testing was on the effectiveness of the PCA.

The Phase 2 study showed the importance of PCA reagent concentration ordose. A different application necessitated an increase dose. PCA at1.54% although effective in an animal open skin wound was not a veryeffective antibacterial reagent on intact human skin. Therefore theconcentration of PCA needed to be increased and could not be increasedwith water, but only be accomplished with the vehicle of 70% isopropylalcohol.

Phase 2 showed that PCA was more effective in the higher concentrationeliminating all bacteria and reducing Propionibacterium acnes to onecolony in two separate instances. It should be noted that the 70%isopropyl alcohol vehicle had little anti-bacterial properties. Thewater was sterile and the alcohol tested negative in vitro. Therefore,they did not contribute other than as a vehicle for topical applicationin this environment.

The interval of 20 minutes was chosen for testing was similar to whatwould be expected if used as a surgical preparation.

The first and 2nd method of harvesting differed. The 2nd wasaccomplished with pressure wiping with the back end of a sterile scalpelso as to maximize the harvest even from sub surface sweat gland and hairfollicles.

The strengths of this study were in the careful attention given to themethod to disadvantage the topical applications effectiveness, yet PCAwas effective and comparable to Betadine tested similarly as Phase 1.The PCA used was a single molecule of 99% pure biochemically synthesizedPCA. The water an and the isopropyl alcohol could be discounted as acontributor to the effect as it contributed nothing to the bactericidaleffect; sterile water and null effect of IPA in vitro testing.

The method used placed a burden on effectiveness of the reagents by theshort time (20 minutes) to act and the 2nd harvest maximizing thepotential yield due to the pressure scraping with back edge of ascalpel.

This study showed the effectiveness of a single reagent, PCA, the commonphytochemical metabolite. It showed that a single concentratedmetabolite working alone is bactericidal on human skin. It showed theimportance of dose variation depending upon the intended topicalapplication; wound versus intact skin. It showed the effectivenessabsent any potential antibacterial boost from the isopropyl alcoholvehicle.

It established the criteria necessary for consideration as a drug;single reagent, known dose effective in two different host environments,topical application route, frequency of application (once), duration (20min) time to gain the intended result.

Phase 3: Another test was conducted where the PCA was dissolved in 15 mlpropylene glycol. The PPH is a skin penetration enhancer and assists indissolving PCA.

In another test, 5 ml of essential oil of Peppermint was added. This hasskin penetration enhancer properties and anti-microbial properties.

Based upon Phase I and II results, it was determined that the dose orconcentration of PCA should be increased. It also was determined that itwould be ideal to have something in the composition that possessed skinpenetration properties. Therefore propylene glycol (PPG) was added.Fasano W J, ten Berge W F, Banton M I, Heneweer M, Moore N P. Dermalpenetration of propylene glycols: measured absorption across humanabdominal skin in vitro and comparison with a QSAR model. Toxicol InVitro. 2011 December; 25(8):1664-70. Then to further the skinpenetration an essential oil was added; i.e. an essence of peppermintoil (EOPO) (Nature oil, 1800 Miller Parkway, Streetsburo, Ohio 44241100% pure [Japan]. Chen J, Jiang Q-D, Wu Y-M, Liu P, Yao J-H, Lu Q,Zhang H, Duan J-A. Potential of Essential Oils as Penetration Enhancersfor Transdermal Administration of Ibuprofen to Treat DysmenorrhoeaMolecules 2015, 20, 18219-18236. Note that both PPG and EOPO are skinpenetration enhancers but also have anti-microbial properties. P. acnesnormally reside deep in the skin's hair follicles and or sebaceousglands. Therefore non-penetration common commercial disinfectants arenot effective as reported in the literature and noted in the Backgroundsection above.

The composition of matter was created when 20 grams of PCA was placed in85 ml 70% isopropyl alcohol making a concentration of PCA 17% (+/−).Then 15 ml propylene glycol and 5 ml of essential oil of peppermint wereadded. All reagents had skin penetration properties. The oppositeshoulder had only the control 70% isopropyl alcohol vehicle applied. Thesubjects volunteered they liked the peppermint smell. The followingsummary is the no growth culture results after treatment.

Reagent Aerobic Anaerobic 17% +/− PCA 10/12 11/12 composition 70% IPA10/12  8/12 aloneThe analysis of the 2 pre PCA treatment subjects that grew subsequentpositive cultures were analyzed as follows:

PCA Pre Treatment Aerobic Species Analysis:

The index subject #18 aerobic growth was too numerous to count. Therewere 2 different species; Staphylococcus epidermidis/hominis andMicrococcus luteus. The Subject #18's post PCA treatment aerobicculture, #20 showed 2 colonies; S. capitis and S. epidermidis. Forreasons unknown, neither of these species was identified in the indexculture, and both are considered non pathogens. The pre-PCA treatmentindex culture on subject #37 had colonies too numerous to count (TNTC)with heavy growth. The colony species were reported as one;Staphylococcus capitus. The Subject #37's post PCA treatment aerobicculture #39, showed one (1) colony growth, but not the former S.capitus, but was identified Staphylococcus epidermidis as the residual.As in prior phases, the residual growth was most often a non pathogen onaerobic culture.

Anaerobic Species Analysis:

The 17% PCA composite solution resulted in 11 of 12 subject's subsequentcultures having no growth. Pre PCA treatment subject #18 had bacteriacolonies too numerous to count (TNTC) on anaerobic culture. There were 4separate colonies with the following species; S. epidermidis (n=3) andPropionibacterium avidium. After this PCA treatment there was noanaerobic growth. Subject #37 index anaerobic culture showed 79colonies. There were 4 prominent colony species; Staphylococcusepidermidis, Staphylococcus capitis/caprae, Staphylococcus capitis andPropionibacterium acnes. The one post PCA culture that showed abacterial colony culture was subject #37 with post PCA treatmentanaerobic culture being #39. There were 2 two colonies of same species;Staphylococcus epidermidis. There were no Propionibacterium acnescolonies. Thus, the results of Phase 3 showed the effectiveness of17%+/− PCA in a composition of matter that had skin penetrationproperties; propylene glycol and essence of peppermint oil.

In Phase IV the method was different from the previous phases:

Method:

The variations in materials and methods in this phase were based uponresults of the previous phases. They included changes in the solution,the method of dissolving the PCA, the timing of skin application, plususing two applications to simulate the present day recommendations ofsurgical skin preparation.

The solution used was 20 mg of ground PCA (Nanjing Zelang MedicalTechnology Co. Ltd.) in 95 ml 91% isopropyl alcohol and 5 ml ofessential oil of peppermint 100% pure. The method placed the 20 grams ofground PCA in sterile container with volume markers. Then added 85 ml of91% isopropyl alcohol; warm and shake. 5 ml of essential oil ofpeppermint was added. A repeat of warming and shaking was instituted.The container was filled to 100 ml, warm and repeated the shaking. Ittook perhaps 15 to 20 minutes for the ground crystals to dissolve.

An area was marked at 4 corners on the anterior axilla area of bothshoulders. There was a topical reagent application. After 5 minutes asecond application was made. The control group was again Chloroprep®.The experimental side was applied with manual motion and pressure on asterile sponge soaked with the PCA solution. The subjects put hands onhead for 10 minutes and then 10 minutes of moving arms around. Culture#1 was harvested with scraping at 20 minutes post 2^(nd) application.Subjects rested their arms at side for 40 minutes which is 60 minutesafter the first application and 40 minutes after culture #1. A secondculture is taken with scraping. The post application cultures were takenof each shoulder skin area with scraping with back side of a sterilesurgical knife blade to maximize the yield.

Results of Phase IV:

The index pre-treatment culture's average colony counts of the 4 groupswere similar.

Aerobic Anaerobic PCA  34.4 average  44.4 average Chloroprep ® 41.75average 49.83 average

The only group showing no growth in all 12 treated subjects was thePCA/peppermint solution treated aerobic group at 20 minutes. Other thanthat group, neither PCA nor Chloroprep® sterilized the skin 100% at 20or 60 minutes. However, Chloroprep® had more “no growth” cultures intotal. The following summary is the no growth cultures results aftertreatment.

Aerobic anaerobic PCA 20 minutes 12/12  4/12 PCA 60 minutes  7/12  6/12Chloroprep ® 20 minutes 10/12  8/12 Chloroprep ® 60 minutes 10/12 10/12

The 20% PCA in 91% IPA and PPO was most effective at 20 minutes onaerobic culture, but not otherwise. Chloroprep® was partially effectiveat 20 minutes and maintained similar effectiveness on aerobic cultureand same effectiveness on anaerobic cultures at 60 minutes. The resultswere different on Chloroprep at 20 minutes from Phase 1 when there wasno growth following this reagent application.

There were 9 cultures that warranted selection for speciesdetermination. #8 pre PCA treatment anaerobic culture had 8 colony countbut zero at 20 and 60 minutes. The interest was to learn what bacterialwere eliminated. There were two species; S. capitis and S. epidermidis.#12 post PCA at 60 minutes showed 1 spreader colony on the plate (SPR)50 colonies. This contrasted with the pre-treatment of 28 colonies. Thespecies found in #12 aerobic were Klebsiella pneumonia/oxytoca andMicrococcus leuteus. These are not common pathogens. #14 pre PCAanaerobic showed 115 colonies. The species were Propionbacterium acnes,S. ludgunessis and Kocuia varians. There was no growth at 20 minutesshowing that potential pathogen P. acnes was eliminated. However therewere 4 aerobic colonies at 60 minutes in #19. They were: Gram NegativeRods: Stenotrophomonas maltophilia: rare pathogen and Stenotrophomonasmaltophilia. The Gram Positive cocci were Kytococcus sedentarius: rareopportunistic pathogen and Micrococcus luteus/lylae: opportunisticpathogen, particularly in hosts with compromised immune systems, such asHIV patients. #19 was a pre PCA anaerobic culture with 39 count showingspecies of P. acnes and S. epidermidis. #21 was the post PCA at 20minutes and showed 1 anaerobic colony of P. acnes, a reduction from 39colonies.

There was culture of Gram Positive Rods with spores identified as:Lysinibacillus sphaericus/fusiform is: rare pathogen. #23 showed nogrowth at 60 minutes for aerobic or anaerobic. Therefore all aerobicbacteria including the one anaerobic colony of P. acnes was gone at 60minutes. #31 aerobic was pre PCA showing 134 colonies. The species wereMicrococcus luteus and Micrococcus luteus/lylae. There was no growth at20 or 60 minutes of these normal flora species. #31 anaerobic was a prePCA showing 28 colonies of Staphylococcus ludgunensis and Bacilluscereus/thuringiensis. There was no growth of either at 20 and 60minutes. #32 was pre Chloroprep with aerobic colony count of 59 with thefollowing species: Micrococcus luteus/lylae, Micrococcus luteus andKocuria Kritinae. There was no colony growth at 20 or 60 minutes. #32was pre Chloroprep anaerobic with 17 colonies with the followingspecies: S. epidermidis and Gemella Bergeri/sanguinis. The posttreatment showed no growth at 20 or 60 minutes. It should be noted inany of the literature or these experiments that re-colonization occurswith skin bacteria, likely in 30 minutes after treatment.

Species Analysis:

The pre-treatment species were predominately saprotrophic or commensalorganisms. Propionibacterium acnes was identified in two subjects. AfterPCA treatment the specific species analysis showed predominatelysaprotrophic or commensal organisms. The potential pathogenPropionibacterium acnes was identified with 19 colonies pre-PCAtreatment in #14 but showed no growth at 20 minutes after PCA treatment.P. acnes was identified pre-PCA treatment in #19 with 19 colonies andpost treatment at 20 minutes there was one remaining colony of P. acnes.However there was no growth at 60 minutes.

Discussion of Phase IV:

This solution of 20 grams of PCA, 75 ml of 91% isopropyl alcohol and 5ml of essence of peppermint oil was effective in removing potentialpathogens, including Propionibacaterium acnes. Only commensal bacteriawith rare pathogenicity remained. The control Cloroprep® did not removedmost of the bacterial colonies, but not all. Many saprotrophic orcommensal organisms were left intact. The PCA composition was not aseffective as Cloroprep® against anaerobes, but where there was post PCAgrowth the species specific analysis showed that Propionibacterium wascontrolled.

A composite of the results based only no growth cultures followingtreatment by each solution are shown in FIG. 42. *Note that the 1% PCAwas not included. The summation percentages of “no growth” are shown inFIG. 43.

Summary of the 4 Phase Testing at Loma Linda:

The questions proposed in the purpose of this proof of principle pilotstudy were answered in the affirmative. The optimal dose of PCA to actas a human skin disinfectant is greater than 10%. There is afacilitating delivery vehicle which is at least 70% isopropyl alcoholwhich allows higher concentrations of PCA to go into solution thanwater, propylene glycol and or essential oils alone. The results wereoptimized by the addition of known skin penetration enhancers; propyleneglycol and essence of peppermint oil. P. acnes normally resides deep inthe skin's hair follicles and or sebaceous glands. Thereforenon-penetration common commercial disinfectants are not effective.

PCA in one or more of these vehicles provided a broad spectrumdisinfectant effect comparable to existing commercial products;isopropyl alcohol, Chloroprep® and Betadine®.

One of two result assessment approaches was considered in this study.One is related to government regulations and is called non-inferioritystatus, or the showing the test article is essentially the same as amarketed product. The reason to choose this method is that the existingmarketed products in the study had side effects, complications, and orhigher cost. D'Agostino Sr R B, Massarol J M, Sullivan L M.Non-inferiority trials: design concepts and issues—the encounters ofacademic consultants in statistics Statist. Med. 2003; 22:169-186.

The other method is to establish clear superiority to an existingproduct. Both were considered in this study. The various PCA solutionsshowed a non-inferiority status to 70% Isopropyl alcohol, Betadine® andChloroprep®. The PCA solutions in this study showed superiority to allbut Chloroprep®. After the first Phase and each subsequent Phaseresults, the materials and methods were modified in attempt to improvethe results of the test reagent PCA. The vehicle was changed from waterto isopropyl alcohol to facilitate an increase dose of PCA. In addition,the vehicle was change to include reagents known to have skinpenetration properties as well as antibiotic properties; i.e. propyleneglycol and essence of peppermint oil. In Phase III the best PCA resultsin both aerobic and anaerobic cultures were obtained with a mixture of17% PCA, propylene glycol and essence of peppermint oil. The anaerobiccultured bacteria are those likely to be below the skin surface andperhaps were affected by the addition of the skin penetrator enhancingreagents. PCA effectiveness was demonstrated in Phase I, even with thelow dose of a 1.24% aqueous solution. This aqueous solution of PCAdecreased the colonies of Propionibacterium acnes that were too numerousto count down to about to 1 and 5 colonies on same subject aftertreatment. Solutions of PCA at 10% or higher removed all pathogens,including P. acnes, but not all bacteria. Based upon the literature andthe results of this study, the goal of complete sterilization of thehuman skin may not be possible, predictable or desirable. In fact,sterilization may not be desirable as it may eliminate bacteria that arebeneficial to skin health and homeostasis. It does seem reasonable toremove all bacteria species that have pathologic potential. The irony ofthis goal is that it is reported that Propionibacterium acnes althoughin some instances has become a pathogen, it also has beneficialproperties secreting a novel antioxidant. PCA which decreased and oreliminated P. acnes in these studies is an anti-oxidant so its presenceas such may substitute for the biological anti-oxidant benefit of P.acnes. To further confuse any such study of this nature, it has beenreported that the amount of bacteria on the skin prior to surgery arenot directly related to surgical site infection. (Cronquist A B, JokobK, Lai L, Latta P D, Larson E L. Relationship between Skin MicrobialCounts and Surgical Site Infection after Neurosurgery. Clin Infect Dis.(2001) 33 (8):1302-1308).

In Phase I, Chloroprep® showed sterilization of the skin in allsubjects. However this was not replicated in Phase IV at 20 minutes orat the extended time of 60 minutes. It should be noted that Saltzman, etal reported in the literature that Chloroprep® had a 7% incidence ofresidual bacteria; Propionibacterium acnes and Staphylococcus aureuspersisted after treatment and his harvest was at a short time in whichthe reagent was still moist and there was not enough time perhaps forrecolonization. (Saltzman M D, Nuber G W, Gryzlo S M, Mareck G S, Koh JL. Efficacy of Surgical Preparation Solutions in Shoulder Surgery. JBone Joint Surg Am, 2009 Aug. 1; 91 (8): 1949-1953). There is no clearreason for the difference in Chloroprep® results in the literature sincethere were many variables in methods and timing of harvesting. There isnot an apparent explanation for the differing results at 20 minutes inPhase I and IV in our study. Yet in Phase IV Chloroprep® was better than20% PCA in 91% isopropyl alcohol and 5 ml of essence of peppermint oilexcept at 20 minute aerobic cultures, which were sterile with the PCAsolution. Note that isopropyl alcohol was not in Phase IV solution.

Another measure of a treatment method would be the effectiveness toreduce bacteria colony counts. This was common with PCA solutions in allphases. A reduction in bacteria is a well-known principle in surgery inorder to reduce the chance of infection. (Anglen J. Perspectives onModern Orthopedics. Wound Irrigation in Musculoskeletal Injury. J AmAcad Ortho Surg. July/August 2001. 9 (4). 219-226).

The invention claimed is:
 1. A method of treating a mammal's skincomprising: treating the mammal's skin with a formulation consistingessentially of: protocatechuic acid or 2,4,6-trihydroxybenzaldehyde, ora mixture or combination thereof, and a carrier; wherein the step oftreating the mammal's skin elicits an effect selected from inhibiting agrowth of pathological microorganisms, eliminating a growth ofpathological microorganisms, and killing pathological microorganismscomprising Propionibacterium acnes, methicillin-resistant Staphylococcusaureus, and Candida albicans, while preserving commensal microorganisms.2. The method of claim 1, wherein the carrier comprises water.
 3. Themethod of claim 1, wherein the carrier is a skin-penetrating carriercomprising a 70% to about 91% solution of isopropyl alcohol.
 4. Themethod of claim 3, wherein the formulation is selected from a sanitizer,a disinfectant, a spray, a rinse, a swab, a salve, a soap, a foam, acream, antibacterial treatments, and a lotion and combinations thereof.5. The method of claim 4, wherein the formulation is a spray or rinse.6. The method of claim 5, further comprising: spraying a surface of themammal's skin to be treated with the formulation; and allowing theformulation to dry, thereby forming a crystalline film of solidprotocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a combinationthereof, wherein the film of solid protocatechuic acid or2,4,6-trihydroxybenzaledehyde or a combination thereof continues to killpersister, pathological microorganisms comprising P. acnes,methicillin-resistant Staphylococcus aureus, and C. albicans.
 7. Themethod of claim 5, further comprising: spraying the surface of themammal's skin to be treated and allowing the formulation to dry a numberof times, thereby forming a crystalline film, wherein the number oftimes is adjusted to control an amount of solid protocatechuic acid or2,4,6-trihydroxybenzaledehyde or a combination thereof within thecrystalline film of solid protocatechuic acid or2,4,6-trihydroxybenzaledhyde or a combination thereof.
 8. The method ofclaim 3, wherein the skin-penetrating carrier further comprises a 5%solution of an essential oil and optionally comprises a 15% solution ofpropylene glycol.
 9. The method of claim 1, wherein the method isperformed before a surgical procedure, during a surgical procedure, orafter a surgical procedure.
 10. The method of claim 9, wherein themammal's skin to be treated is on the hands of a surgeon to perform thesurgical procedure.
 11. The method of claim 9, wherein the mammal's skinto be treated is on a patient to undergo the surgical procedure.
 12. Themethod of claim 1, wherein the method is performed before a dentalprocedure, during a dental procedure, or after a dental procedure.